H. Cai et al., Protein Pept Lett (2017).
Propeptides have been demonstrated to influence many properties of their mature proteins as intramolecular chaperones. In our study, the role of pentapeptide prosequence of Thermomyces lanuginosus lipase (TLL) was determined through its effect on the recombinant expression of TLL in P. pastoris. The result showed that the average lipase activity of recombinant strains GS-pTL reached 434.32 U/mL, higher than that of GS-TL 377.71 U/mL, indicating that the propeptide "SPIRR-" sequence is beneficial for enhancing TLL expression. In addition, rational design and site-directed mutation were performed to increase the propeptide's hydrophobicity to explore its possible function mechanism. The fermentation result of the recombinant strains with modified propeptide showed that the extracellular lipase activity of GS-pTL-VP variant reached 483.29 U/mL, increasing by 11.27% compared with that of GS-pTL (434.32 U/mL). Therefore, the increase of hydrophobicity in propeptide was considered to be advantageous to enhance the expression of lipase TLL, possibly through assisting the protein nucleation as a hydrophobic scaffold. This study would provide new insight into the role of short propeptide in protein expression and structure folding.
C. Aponte-Santamaria et al., Sci Rep 7 4016 (2017).
Regulation of aquaporins is a key process of living organisms to counteract sudden osmotic changes. Aqy1, which is a water transporting aquaporin of the yeast Pichia pastoris, is suggested to be gated by chemo-mechanical stimuli as a protective regulatory-response against rapid freezing. Here, we tested the influence of temperature by determining the X-ray structure of Aqy1 at room temperature (RT) at 1.3 A resolution, and by exploring the structural dynamics of Aqy1 during freezing through molecular dynamics simulations. At ambient temperature and in a lipid bilayer, Aqy1 adopts a closed conformation that is globally better described by the RT than by the low-temperature (LT) crystal structure. Locally, for the blocking-residue Tyr31 and the water molecules inside the pore, both LT and RT data sets are consistent with the positions observed in the simulations at room-temperature. Moreover, as the temperature was lowered, Tyr31 adopted a conformation that more effectively blocked the channel, and its motion was accompanied by a temperature-driven rearrangement of the water molecules inside the channel. We therefore speculate that temperature drives Aqy1 from a loosely- to a tightly-blocked state. This analysis provides high-resolution structural evidence of the influence of temperature on membrane-transport channels.
H. Kerr et al., J Biol Chem (2017).
Spontaneous activation enables the complement system to respond very rapidly to diverse threats. Activation is efficiently suppressed by complement factor H (CFH) on self surfaces but not foreign surfaces. The surface selectivity of CFH, a soluble protein containing 20 CCP modules (CCPs 1-20), may be compromised by disease-linked mutations. Which of the several functions of CFH drives its self-surface selectivity remains unknown. We made human CFH mutants in Pichia pastoris. In benchmark studies of CCP-1 variants, recombinant I62-CFH (protective against age-related macular degeneration) and V62-CFH functioned equivalently, matching or outperforming plasma-derived CFH while R53H-CFH, linked to atypical haemolytic uraemic syndrome (aHUS), was defective in C3bBb decay-accelerating activity (DAA) and factor I-cofactor activity (CA). The aHUS-linked CCP-19 mutant D1119G-CFH had virtually no CA on (self-like) sheep erythrocytes (ES) but retained measurable DAA. The aHUS-linked CCP-20 mutant S1191L/V1197A-CFH (LA-CFH) had dramatically reduced CA on ES but was less compromised with respect to DAA. D1119G-CFH and LA-CFH performed poorly at preventing complement-mediated haemolysis of ES. PspCN, a CFH-binding Streptococcus pneumoniae protein domain, increases accessibility of CCPs 19-20. It did not improve the DAA of any CFH variant on ES although it enhanced DAA in surface plasmon-resonance studies. Conversely, PspCN boosted the CA on ES of I62-CFH, R53H-FH and LA-CFH. PspCN also enhanced haemolysis protection by I62-CFH and LA-CFH. We concluded that CCPs 19-20 are critical for efficient CA on self-surfaces but less important for DAA. By exposing CCPs 19-20 and enhancing CA on self-surfaces, PspCN might therapeutically reverse deficiencies in some CFH variants.
T. Shang et al., BMC Biotechnol 17 55 (2017).
BACKGROUND: Xylanase degrades xylan into monomers of various sizes by catalyzing the endohydrolysis of the 1,4-beta-D-xylosidic linkage randomly, possessing potential in wide industrial applications. Most of xylanases are susceptible to be inactive when suffering high temperature and high alkaline process. Therefore, it is necessary to develop a high amount of effective thermoalkaliphilic xylanases. This study aims to enhance thermoalkaliphilic xylanase production in Pichia pastoris through fermentation parameters optimization and novel efficient fed-batch strategy in high cell-density fermentation.
RESULTS: Recombinant xylanase activity increased 12.2%, 7.4%, 12.0% and 9.9% by supplementing the Pichia pastoris culture with 20 g/L wheat bran, 5 mg/L L-histidine, 10 mg/L L-tryptophan and 10 mg/L L-methionine in shake flasks, respectively. Investigation of nutritional fermentation parameters, non-nutritional fermentation parameters and feeding strategies in 1 L bioreactor and 1 L shake flask revealed that glycerol and methanol feeding strategies were the critical factors for high cell density and xylanase activity. In 50 L bioreactor, a novel glycerol feeding strategy and a four-stage methanol feeding strategy with a stepwise increase in feeding rate were developed to enhance recombinant xylanase production. In the initial 72 h of methanol induction, the linear dependence of xylanase activity on methanol intake was observed (R2 = 0.9726). The maximum xylanase activity was predicted to be 591.2 U/mL, while the actual maximum xylanase activity was 560.7 U/mL, which was 7.05 times of that in shake flask. Recombinant xylanase retained 82.5% of its initial activity after pre-incubation at 80 degrees C for 50 min (pH 8.0), and it exhibited excellent stability in the broad temperature (60-80 degrees C) and pH (pH 8.0-11.0) ranges.
CONCLUSIONS: Efficient glycerol and methanol fed-batch strategies resulting in desired cell density and xylanase activity should be applied in other P. pastoris fermentation for other recombinant proteins production. Recombinant xylanases with high pH- and thermal-stability showed potential in various industrial applications.
C. Walther, A. Durauer, Biotechnol J (2017).
Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris are the standard platforms for biopharmaceutical production with 40% of all between 2010 to 2014 approved protein drugs produced in those microbial hosts. Typically, products overexpressed E. coli and S. cerevisiae remain in the cytosol or are secreted into the periplasm. Consequently, efficient cell disruption is essential for high product recovery during microbial production. Process development platforms at microscale are essential to shorten time to market. While high-pressure homogenization is the industry standard for cell disruption at large scale this method is not practicable for experiments in microscale. This review describes microscale methods for cell disruption at scales as low as 200 microL. Strategies for automation, parallelization and miniaturization, as well as comparability of the results at this scale to high pressure homogenization are considered as those criteria decide which methods are most suited for scale down. Those aspects are discussed in detail for protein overexpression in E. coli and yeast but also the relevance for alternative products and host such as microalgae are taken into account. The authors conclude that bead milling is the best comparable microscale method to large scale high-pressure homogenization and therefore the most suitable technique for automated process development of microbial hosts with the exception of pDNA production.
L. Morales-Quintana et al., Carbohydr Res 448 57 (2017).
The fungus Penicillium purpurogenum degrades plant cell walls by the action of cellulolytic, xylanolytic and pectinolytic enzymes. The alpha-D-galactosidase is one of the enzymes which may act on pectin degradation. This enzyme has several biotechnological and medical applications. The aim of this work was to better understand the molecular mechanism of alpha-D-galactosidase from P. purpurogenum (GALP1). For this purpose, a gene coding for the enzyme was identified from the fungal genome and heterologously expressed in Pichia pastoris. The enzyme belongs to glycosyl hydrolase family 27. The protein of 435 amino acids has an optimum pH and temperature for activity of 5.0 and 50 degrees C, respectively. The KM for p-nitrophenyl-alpha-D-galactopyranoside (GalalphapNP) is 0.138 mM. The enzyme is inhibited by GalalphapNP at concentrations higher than 1 mM, and by the product galactose. A kinetic analysis of product inhibition shows that it is of mixed type, suggesting the presence of an additional binding site in the enzyme. To confirm this hypothesis, a structural model for GALP1 was built by comparative modelling methodology, which was validated and refined by molecular dynamics simulation. The data suggest that galactose may bind to an enzyme alternative pocket promoting structural changes of the active site, thus explaining its inhibitory effect. In silico site-directed mutagenesis experiments highlighted key residues involved in the maintenance of the alternative binding site, and their mutations for Ala predict the formation of proteins which should not be inhibited by galactose. The availability of an alpha-galactosidase with different kinetic properties to the existent proteins may be of interest for biotechnological applications.
F. Wang et al., Vet Microbiol 205 124 (2017).
Linear DNA vaccines provide effective vaccination. However, their application is limited by high cost and small scale of the conventional polymerase chain reaction (PCR) generally used to obtain sufficient amounts of DNA effective against epidemic diseases. In this study, a two-step, large-scale PCR was established using a low-cost DNA polymerase, RKOD, expressed in Pichia pastoris. Two linear DNA vaccines encoding influenza H1N1 hemagglutinin (HA) 1, LEC-HA, and PTO-LEC-HA (with phosphorothioate-modified primers), were produced by the two-step PCR. Protective effects of the vaccines were evaluated in a mouse model. BALB/c mice were immunized three times with the vaccines or a control DNA fragment. All immunized animals were challenged by intranasal administration of a lethal dose of influenza H1N1 virus 2 weeks after the last immunization. Sera of the immunized animals were tested for the presence of HA-specific antibodies, and the total IFN-gamma responses induced by linear DNA vaccines were measured. The results showed that the DNA vaccines but not the control DNA induced strong antibody and IFN-gamma responses. Additionally, the PTO-LEC-HA vaccine effectively protected the mice against the lethal homologous mouse-adapted virus, with a survival rate of 100% versus 70% in the LEC-HA-vaccinated group, showing that the PTO-LEC-HA vaccine was more effective than LEC-HA. In conclusion, the results indicated that the linear H1N1 HA-coding DNA vaccines induced significant immune responses and protected mice against a lethal virus challenge. Thus, the low-cost, two-step, large-scale PCR can be considered a potential tool for rapid manufacturing of linear DNA vaccines against emerging infectious diseases.
J. Y. Lee et al., Protein Expr Purif (2017).
Pichia pastoris is a highly successful recombinant protein expression system due to its ability to quickly generate large quantities of recombinant proteins in simple media. P. pastoris has been used to successfully generate milligram quantities of many important human membrane proteins, including G-protein coupled receptors, ion channels, and transporters, which are becoming increasingly important therapeutic targets. Despite these successes, protein expression in P. pastoris is still cumbersome due to a need to change growth media from glycerol media to methanol induction media, which minimizes inhibition of the AOX1 promoter by residual glycerol. Taking advantage of this behavior of the AOX1 promoter, we developed Buffered extra-YNB Glycerol Methanol (BYGM) auto-induction media (100 mM potassium phosphate pH 6.0, 2.68% w/v YNB, 0.4% v/v glycerol, 0.5% v/v methanol, and 8 x 10-5% w/v biotin) which not only simplified the protein expression process, but also optimized protein expression levels in P. pastoris. We successfully used this auto-induction method to overexpress the target in both MutS and Mut+ strains. Moreover, we show that this method can facilitate screening high-expressing clones, as well as enable parallel protein production in P. pastoris.
X. Liu et al., J Food Sci (2017).
Xylanase inhibitor proteins (XIPs) were regarded to inhibit the activity of xylanases during baking and gluten-starch separation processes. To avoid the inhibition to xylanases, it is necessary to define the conditions under which the inhibition takes place. In this study, we cloned the XIP gene from 2 different variety of Triticum aestivum, that is, Zhengmai 9023 and Zhengmai 366, and investigated the properties of XIP protein expressed by Pichia pastoris. The results showed that the 2 XIP genes (xip-9023 and xip-366) were highly homologous with only 3 nucleotide differences. XIP-9023 showed the optimal inhibition pH and temperature were 7 degrees C and 40 degrees C, respectively. Inhibition of xylanase by XIP-9023 reached the maximum in 40 min. At 50% inhibition of xylanase, the molar ratio of inhibitor: xylanase was 26:1. XIP-9023 was active to various fungal xylanases tested as well as to a bacterial xylanase produced by Paenibacillus sp. isolated from cow rumen.
J. Zhang, T. Liu, Front Microbiol 8 963 (2017).
Pichia pastoris is a good model for pexophagy research owing to its diverse pexophagy modes (macropexophagy and micropexophagy) exhibited during carbon-source shift from methanol to other carbon sources. The critical condition that triggers activation of macropexophagy and micropexophagy is important for clarifying the P. pastoris pexophagy mechanism and human peroxisomal disorders. In this study, the pexophagy modes of P. pastoris were confirmed by green fluorescent protein expression and alcohol oxidase and formate dehydrogenase activities. Furthermore, intracellular energy charge (EC) was found to be a determinant of pexophagy activation. During methanol induction, the EC was about 0.5. And the final EC value was related to the pexophagy mode when carbon source switched from methanol to others. Macropexophagy and micropexophagy occurred when the EC increased to 0.6-0.75 and above 0.75, respectively. Thus, different EC values were considered as the important factor to trigger different pexophagy modes in P. pastoris. The results obtained in this study could help in achieving better control of the pexophagy modes to study the pexophagy mechanism.
F. Roohvand, M. Shokri, M. Abdollahpour-Alitappeh, P. Ehsani, Expert Opin Ther Pat 1 (2017).
INTRODUCTION: Yeasts, as Eukaryotes, offer unique features for ease of growth and genetic manipulation possibilities, making it an exceptional microbial host. Areas covered: This review provides general and patent-oriented insights into production of biopharmaceuticals by yeasts. Patents, wherever possible, were correlated to the original or review articles. The review describes applications of major GRAS (generally regarded as safe) yeasts for the production of therapeutic proteins and subunit vaccines; additionally, immunomodulatory properties of yeast cell wall components were reviewed for use of whole yeast cells as a new vaccine platform. The second part of the review will discuss yeast- humanization strategies and innovative applications. Expert opinion: Biomedical applications of yeasts were initiated by utilization of Saccharomyces cerevisiae, for production of leavened (fermented) products, and advanced to serve to produce biopharmaceuticals. Higher biomass production and expression/secretion yields, more similarity of glycosylation patterns to mammals and possibility of host-improvement strategies through application of synthetic biology might enhance selection of Pichia pastoris (instead of S. cerevisiae) as a host for production of biopharmaceutical in future. Immunomodulatory properties of yeast cell wall beta-glucans and possibility of intracellular expression of heterologous pathogen/tumor antigens in yeast cells have expanded their application as a new platform, 'Whole Yeast Vaccines'.
L. A. Lima et al., Biopolymers (2017).
One of the roadblocks towards the practical use of antimicrobial peptides for medical use is their relatively high cost when synthesized chemically. Effective recombinant production has only been successful in some cases, such as the previously reported production in Pichia pastoris of the antimicrobial plectasin derivative peptide NZ2114. The same production host has also been used extensively to produce so-called protein-polymers: sequences that consist of repetitions of simple amino acid motifs found in structural proteins such as collagen and elastin, and that can be designed to self-assemble in micelles, fibers and hydrogels. With the eventual goal of producing recombinant biomaterials such as antimicrobial protein polymer, we here explore the secreted production in Pichia pastoris of a fusion of NZ2114 with a hydrophilic random coil protein polymer CP4 . The intact NZ2114-CP4 fusion copolymer was produced with a yield of purified protein on the order of 1 g.L-1 supernatant. We find that purified NZ2114-CP4 has an activity against clinical strain MRSA, but very much lower than activity of chemically synthesized NZ2114. We conclude that possibly, the activity of NZ2114 is impaired by the C-terminal attachment to the protein polymer chain, but other reasons for the low activity cannot yet be excluded either. This article is protected by copyright. All rights reserved.
Y. Zhang et al., Protein Expr Purif 136 20 (2017).
The Botrytis cinerea BcSpl1 protein is a member of the cerato-platanin family, and consists of 137 amino acids and two disulfide bridges. This protein induces the onset of necrosis in infiltrated plant hosts. Recombinant BcSpl1 proteins produced in Pichia pastoris (pBcSpl1) and Escherichia coli (eBcSpl1) were initially compared regarding their abilities to induce necrosis and systemic acquired resistance (SAR). The pBcSpl1 and eBcSpl1 treatments led to the development of necrotic lesions on tomato leaves, and provided tomato plants with SAR to B. cinerea. The lesion area of leaves infiltrated with the BcSpl1 proteins decreased by 22.7% (pBcSpl1) and 21.8% (eBcSpl1). Additionally, eBcSpl1 up-regulated the expression levels of some defense-related genes, including PR-1a, prosystemin, PI I, and PI II, as well as SIPK and TPK1b, which encode two protein kinases. Furthermore, eBcSpl1 exhibited chitin-binding properties. Our data revealed that the E. coli expression system produces higher BcSpl1 yields than the P. pastoris system. This high-yield expression of BcSpl1 may be relevant for future large-scale applications of this elicitor to improve crop production.
T. Linder, Curr Microbiol (2017).
The methylotrophic yeast Komagataella pastoris (syn. Pichia pastoris) is one of the few known yeasts that can utilize sulfamate ([Formula: see text]) as a sulfur source. The biochemical pathway responsible for the catabolism of sulfamate has yet to be identified. The present study sought to investigate whether sulfamate catabolism proceeds through either of the inorganic sulfur intermediates sulfate ([Formula: see text]) or sulfite ([Formula: see text]) before its assimilation and subsequent incorporation into sulfur-containing amino acids and their derivatives. Two key genes in the K. pastoris inorganic sulfur assimilation pathway were deleted separately and the ability of each deletion mutant to utilize sulfamate and other selected sulfur sources was studied. Deletion of the MET3 gene (which encodes the enzyme ATP sulfurylase) did not affect growth on L-methionine, sulfite, methanesulfonate, or taurine but completely abolished growth on sulfate, methyl sulfate and sulfamate. Deletion of the MET5 gene (which encodes the beta subunit of the enzyme sulfite reductase) abolished growth on all tested sulfur sources except L-methionine. These results suggest that the catabolism of sulfamate proceeds through a sulfate intermediate before its assimilation.
M. Avelar et al., Protein Expr Purif 136 14 (2017).
In this work we communicate the heterologous expression of a laccase from Coriolopsis gallica in Pichia pastoris. This enzyme has been reported to efficiently degrade a variety of pollutants such as industrial dyes. The expression strategy included using a previously reported modified alpha-factor preproleader for enhanced secretion and pAOX1, a methanol-responsive promoter. Methanol concentration, copper salts concentration and temperature were varied in order to enhance laccase expression in this heterologous system. A volumetric activity of 250 U/L was achieved after 12-day culture in Fernbach flasks. The protein was recovered from the supernatant and purified, obtaining a preparation with 90% electrophoretic purity. The catalytic constants of the recombinant enzyme are almost identical to the fungal enzyme, thus rendering this system a useful tool for protein engineering of laccase from C. gallica.
H. Wang et al., Protein Expr Purif (2017).
A GH11 xylanase gene (xyn11-1) cloned from saline-alkali soil was successfully expressed in Pichia pastoris GS115. The purified recombinant Xyn11-1 showed its maximal activity at pH 6.0, and retained more than 60.4% of activity at pH 10.0, with good pH stability. Its optimal temperature was 50 degrees C and it was stable after incubation for 1 h at 30 degrees C. Furthermore, Xyn11-1 was highly salt-tolerant, retaining more than 77.4% of activity in the presence of 0.25-4 M NaCl and displaying more than 47.2% relative activity after being incubated in the presence of 5 M NaCl at 37 degrees C for 10 min. In addition, 5 mM beta-Mercaptoethanol, Cu2+, Co2+, and Mn2+ increased the xylanase activity by 22.3%, 8.8%, 7.1%, and 4.4%, respectively. Significantly, 93.4% and 59.8% of the optimal activity was retained in the presence of 2% and 10% (v/v) ethanol, respectively. Under optimal conditions, the Km,Vmax, and Kcat value of Xyn11-1 for beechwood xylan were 3.7 mg ml-1, 101.0 mumol min-1 mg-1 and 42.1 s-1, respectively. Xyn11-1 is a strict endo-beta-1,4-xylanase, its main enzymatic products being xylotetraose and xylopentaose. Xyn11-1 is the first reported GH11 xylanase isolated from saline-alkali soil, and has excellent tolerance of high pH, high salt concentrations and ethanol, which indicates its great potential for basic research and industrial applications.
C. Gamerith et al., Front Microbiol 8 938 (2017).
To study hydrolysis of aromatic and aliphatic polyesters cutinase 1 from Thermobifida cellulosilytica (Thc_Cut1) was expressed in P. pastoris. No significant differences between the expression of native Thc_Cut1 and of two glycosylation site knock out mutants (Thc_Cut1_koAsn and Thc_Cut1_koST) concerning the total extracellular protein concentration and volumetric activity were observed. Hydrolysis of poly(ethylene terephthalate) (PET) was shown for all three enzymes based on quantification of released products by HPLC and similar concentrations of released terephthalic acid (TPA) and mono(2-hydroxyethyl) terephthalate (MHET) were detected for all enzymes. Both tested aliphatic polyesters poly(butylene succinate) (PBS) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) were hydrolyzed by Thc_Cut1 and Thc_Cut1_koST, although PBS was hydrolyzed to significantly higher extent than PHBV. These findings were also confirmed via quartz crystal microbalance (QCM) analysis; for PHBV only a small mass change was observed while the mass of PBS thin films decreased by 93% upon enzymatic hydrolysis with Thc_Cut1. Although both enzymes led to similar concentrations of released products upon hydrolysis of PET and PHBV, Thc_Cut1_koST was found to be significantly more active on PBS than the native Thc_Cut1. Hydrolysis of PBS films by Thc_Cut1 and Thc_Cut1_koST was followed by weight loss and scanning electron microscopy (SEM). Within 96 h of hydrolysis up to 92 and 41% of weight loss were detected with Thc_Cut1_koST and Thc_Cut1, respectively. Furthermore, SEM characterization of PBS films clearly showed that enzyme tretment resulted in morphological changes of the film surface.
M. O. Betancur et al., Microb Cell Fact 16 99 (2017).
BACKGROUND: A commonly used approach to improve recombinant protein production is to increase the levels of expression by providing extra-copies of a heterologous gene. In Komagataella phaffii (Pichia pastoris) this is usually accomplished by transforming cells with an expression vector carrying a drug-resistance marker following a screening for multicopy clones on plates with increasingly higher concentrations of an antibiotic. Alternatively, defective auxotrophic markers can be used for the same purpose. These markers are generally transcriptionally impaired genes lacking most of the promoter region. Among the defective markers commonly used in Saccharomyces cerevisiae is leu2-d, an allele of LEU2 which is involved in leucine metabolism. Cells transformed with this marker can recover prototrophy when they carry multiple copies of leu2-d in order to compensate the poor transcription from this defective allele.
RESULTS: A K. phaffii strain auxotrophic for leucine (M12) was constructed by disrupting endogenous LEU2. The resulting strain was successfully transformed with a vector carrying leu2-d and an EGFP (enhanced green fluorescent protein) reporter gene. Vector copy numbers were determined from selected clones which grew to different colony sizes on transformation plates. A direct correlation was observed between colony size, number of integrated vectors and EGFP production. By using this approach we were able to isolate genetically stable clones bearing as many as 20 integrated copies of the vector and with no significant effects on cell growth.
CONCLUSIONS: In this work we have successfully developed a genetic system based on a defective auxotrophic which can be applied to improve heterologous protein production in K. phaffii. The system comprises a K. phaffii leu2 strain and an expression vector carrying the defective leu2-d marker which allowed the isolation of multicopy clones after a single transformation step. Because a linear correlation was observed between copy number and heterologous protein production, this system may provide a simple approach to improve recombinant protein productivity in K. phaffii.
P. Phadtare, S. Joshi, T. Satyanarayana, Int J Biol Macromol 104 107 (2017).
Codon adaptation index (CAI) of a 1263bp long endoglucanase encoding gene from the thermophilic mould Myceliopthora thermophile BJA has been improved from 0.44 to 0.76 by in vitro gene synthesis. The codon optimized endoglucanase gene (Mt-egl) has been constitutively expressed in Pichia pastoris under the regulation of GAP promoter. Recombinant endoglucanase (rMt-egl), purified by size exclusion chromatography, has been confirmed to be a monomeric protein of approximately 47kDa. rMt-egl is optimally active at pH 10 and 50 degrees C, displaying stability in broad pH and temperature ranges, with a t1/2 of 60 and 15min at 90 and 100 degrees C, respectively. This retained approximately 70% of activity after 3h incubation at pH 5-12. The Km, Vmax, kcat and kcat/Km of rMt-egl were 5mgmL-1, 20mumolesmin-1mg-1, 1.02x103s-1 and 204s-1mg-1mL-1, respectively. Homology modeling and bioinformatics analysis confirmed catalytically important role of glutamate 234 and 344. rMt-egl released high amounts of reducing sugars from wheat bran and corn cobs (421 and 382mgg-1), thus making it a useful biocatalyst for producing bioethanol and fine chemicals from agro-residues.
M. A. Abd Wahid et al., Biosci Biotechnol Biochem 1 (2017).
The natural coagulant Moringa oleifera lectin (MoL) as cationic protein is a promising candidate in coagulation process of water treatment plant. Introducing the gene encoding MoL into a host, Pichia pastoris, to secrete soluble recombinant protein is assessed in this study. Initial screening using PCR confirmed the insertion of MoL gene, and SDS-PAGE analysis detected the MoL protein at 8 kDa. Cultured optimization showed the highest MoL protein at 520 mg/L was observed at 28 degrees C for 144 h of culturing by induction in 1% methanol. Approximately, 0.40 mg/mL of recombinant MoL protein showed 95 +/- 2% turbidity removal of 1% kaolin suspension. In 0.1% kaolin suspension, the concentration of MoL at 10 mug/mL exhibits the highest turbidity reduction at 68 +/- 1%. Thus, recombinant MoL protein from P. pastoris is an effective coagulant for water treatment.
H. Qiao et al., Protein Expr Purif (2017).
Relatively poor heterologous protein yields have limited the frequency of Galactomyces geotrichum lipase I (GGl I) efficacy trials. To address this, we have redesigned the GGl I gene to preferentially match codon frequencies of Pichia pastoris (P. pastoris) while retaining the same amino acid sequence. The wild type and codon optimised GGl I (GGl I-wt and GGl I-op) were synthesised and cloned into pPICZalphaA with an N-terminal 6 x His tag sequence and expressed in P. pastoris X 33. The hydrolytic activity of GGl I-op was 150 U/mL, whereas the activity of the GGl I-wt could not be detected. GGl I-op recombinant proteins were purified by Ni-affinity chromatography and then characterised. The identity and purity of GGl I were confirmed by SDS-PAGE, MALDI-TOF mass spectrometry and Western blot analysis. Enzymatic deglycosylation was used to show that the lipase is a glycosylated protein, containing approximately 10% sugar. The molecular weight (MW) of the GGl I secreted by recombinant P. pastoris was approximated at 63 kDa. The optimum pH and temperature of the recombinant lipase were 8.0 and 35 degrees C, respectively. The enzyme was active over a broad pH range (7.0-9.0) and temperature range (20 degrees C-45 degrees C). The lipase showed high activity toward medium- and long-chain fatty acid methyl esters (C8-C16) and retained much of its activity in the present of Tween-80 and Trition X-100. Lipase activity was stimulated by Mg2+, Ca2+, Mn2+ and Cu2+ and inhibited by Fe2+, Fe3+, Zn2+ and Co2+. This lipase may prove useful to the detergent industry and in organic synthesis reactions.
M. Schwaiger et al., Anal Chem (2017).
In this work, simultaneous targeted metabolic profiling by isotope dilution and non-targeted fingerprinting is proposed for cancer cell studies. The novel streamlined metabolomics workflow was established using anion-exchange chromatography (IC) coupled to high resolution mass spectrometry (MS). The separation time of strong anion-exchange (2 mm column, flow rate 380 microL min-1, injection volume 5 microL) could be decreased to 25 min for a target list comprising organic acids, sugars, sugar phosphates and nucleotides. Internal standardization by fully 13C labeled Pichia pastoris extracts enabled absolute quantification of the primary metabolites in adherent cancer cell models. Limits of detection (LODs) in the low nM range and excellent intermediate precisions of the isotopologue ratios (on average < 5% N=5 over 40 hours) were observed. As a result of internal standardization, linear dynamic ranges over 4 orders of magnitude (5 nM - 50 microM, R2 > 0.99) were obtained. Experiments on drug-sensitive versus resistant SW480 cancer cells showed the feasibility of merging analytical tasks into one analytical run. Comparing fingerprinting with and without internal standard proved that the presence of the 13C labeled yeast extract required for absolute quantification was not detrimental to non-targeted data evaluation. Several interesting metabolites were discovered by accurate mass and comparing MS2 spectra (acquired in ddMS2 mode) with spectral libraries. Significant differences revealed distinct metabolic phenotypes of drug-sensitive and resistant SW480 cells.
D. Zhang et al., J Agric Food Chem 65 5041 (2017).
A novel alpha-amylase, Amy13A, that consists of these domains was identified in Talaromyces leycettanus JCM12802: catalytic TIM-barrel fold, domain B, domain C, Thr/Ser-rich linker region, and C-terminal CBM20 domain. The wild type and three mutant enzymes were then expressed in Pichia pastoris GS115 to identify the roles of linker length (Amy13A21 and Amy13A33) and CBM20 (Amy13A-CBM) in catalysis. All enzymes had similar enzymatic properties, exhibiting optimal activities at pH 4.5-5.0 and 55-60 degrees C, but varied in catalytic performance. When using soluble starch as the substrate, Amy13A21 and Amy13A33 showed specific activities (926.3 and 537.8 units/mg, respectively, vs 252.1 units/mg) and catalytic efficiencies (kcat/Km, 25.7 and 22.0 mL s-1 mg-1, respectively, vs 15.4 mL s-1 mg-1) higher than those of the wild type, while Amy13A-CBM performed worse during catalysis. This study reveals the key roles of the CBM and linker length in the catalysis of GH13 alpha-amylase.
S. Aich et al., Biotechnol Biofuels 10 135 (2017).
BACKGROUND: Bipolaris sorokiniana is a filamentous fungus that causes spot blotch disease in cereals like wheat and has severe economic consequences. However, information on the identities and role of the cell wall-degrading enzymes (CWDE) in B. sorokiniana is very limited. Several fungi produce CWDE like glycosyl hydrolases (GHs) that help in host cell invasion. To understand the role of these CWDE in B. sorokiniana, the first step is to identify and annotate all possible genes of the GH families like GH3, GH6, GH7, GH45 and AA9 and then characterize them biochemically.
RESULTS: We confirmed and annotated the homologs of GH3, GH6, GH7, GH45 and AA9 enzymes in the B. sorokiniana genome using the sequence and domain features of these families. Quantitative real-time PCR analyses of these homologs revealed that the transcripts of the BsGH7-3 (3rd homolog of the GH 7 family in B. sorokiniana) were most abundant. BsGH7-3, the gene of BsGH7-3, was thus cloned into pPICZalphaC Pichia pastoris vector and expressed in X33 P. pastoris host to be characterized. BsGH7-3 enzyme showed a temperature optimum of 60 degrees C and a pHopt of 8.1. BsGH7-3 was identified to be an endoglucanase based on its broad substrate specificity and structural comparisons with other such endoglucanases. BsGH7-3 has a very long half-life and retains 100% activity even in the presence of 4 M NaCl, 4 M KCl and 20% (v/v) ionic liquids. The enzyme activity is stimulated up to fivefold in the presence of Mn+2 and Fe+2 without any deleterious effects on enzyme thermostability.
CONCLUSIONS: Here we reanalysed the B. sorokiniana genome and selected one GH7 enzyme for further characterization. The present work demonstrates that BsGH7-3 is an endoglucanase with a long half-life and no loss in activity in the presence of denaturants like salt and ionic liquids, and lays the foundation towards exploring the Bipolaris genome for other cell wall-degrading enzymes.
Z. Dai et al., Bioresour Technol (2017).
The aim of this study was to metabolically construct Saccharomyces cerevisiae for achievement of direct methanol utilization and value added product (mainly pyruvate) production. After successful integration of methanol oxidation pathway originated from Pichia pastoris into the chromosome of S. cerevisiae, the recombinant showed 1.04g/L consumption of methanol and 3.13% increase of cell growth (OD600) when using methanol as the sole carbon source. Moreover, 0.26g/L of pyruvate was detected in the fermentation broth. The supplementation of 1g/L yeast extract could further improve cell growth with increase of 11.70% and methanol consumption to 2.35g/L. This represents the first genetically modified non-methylotrophic eukaryotic microbe for direct methanol utilization and would be of great value concerning the development of biotechnological processes.
X. Ma et al., Enzyme Microb Technol 103 34 (2017).
Laccases are oxidoreductase catalyze the oxidation of a wide range of substrates with oxygen as the electron acceptor. This report was aimed to the high-level expression of a laccase, CueO from Escherichia coli K12 in Pichia pastoris GS115 and its application on decolorization of synthetic dyes. The yacK gene coding CueO was cloned into an expression vector of Pichia pastoris, pHBM905BDM and expressed in a secretory form with Pichia pastoris GS115 as the host. The yield of the recombinant protein was 556mg/L with high-density fermentation and the enzyme activity was about 41,000U/L. The recombinant laccase was purified and characterized. Its optimum pH and temperature was 3.0 and 55 degrees C with 2, 2'-azino-bis-(3-ethylbenzothazoline-6-sulfonic acid) (ABTS) as the substrate, respectively. This recombinant protein was thermostable and its half life at 70 degrees C was about 60min. In the presence of natural redox mediator acetosyringone, the purified recombinant laccase decolorized 98.1% and 98.5% of Congo red, malachite green, respectively. It also decolorized 90.03% of Remazol brilliant blue R without this mediator. In addition, this enzyme was applied on the decolorization of wastewater from a textile printing factory and showed an obvious bleaching effect.
Z. Wang et al., J Immunol Methods (2017).
Ontak(R) is a FDA-approved diphtheria toxin-based recombinant fusion toxin for treatment of human CD25+ cutaneous T cell lymphoma (CTCL). However, it has been discontinued clinically due to the production issue related to the bacterial expression system with difficult purification. Recently we have developed monovalent and bivalent human IL-2 fusion toxins targeting human CD25+ cells using advanced unique diphtheria toxin resistant yeast Pichia Pastoris expression system. In vitro efficacy characterization using human CD25+ HUT102/6TG cells demonstrated that both monovalent and bivalent isoforms are potent and the bivalent isoform is approximately two logs more potent than the monovalent isoform. In this study, we further assessed the in vivo efficacy of the human IL-2 fusion toxins using human CD25+ HUT102/6TG tumor-bearing NSG mouse model. The data demonstrated that both monovalent and bivalent human IL-2 fusion toxins significantly prolonged the survival of the human CD25+ tumor-bearing NSG mice in a dose-dependent manner. Then we further assessed the residual tumor cells from the HUT102/6TG tumor-bearing NSG mice using the residual tumor cell bearing NSG mouse model. The results demonstrated that the residual tumor cells were still sensitive to the continual treatment with the human IL-2 fusion toxin. This yeast-expressed human IL-2 fusion toxin will be a promising candidate to replace the clinically discontinued Ontak(R).
R. Ye et al., Bioresour Bioprocess 4 22 (2017).
BACKGROUND: Pichia pastoris is one of the most important cell factories for production of industrial enzymes and heterogenous proteins. The genome-scale metabolic model of high quality is crucial for comprehensive understanding of the P. pastoris metabolism.
METHODS: In this paper, we upgraded P. pastoris genome-scale metabolic model based on the combination of latest genome annotations and literatures. Then the performance of the new model was evaluated using the Cobra Toolbox v2.0.
RESULTS: Compared with the recently published model iMT1026, the reaction number in the new model iRY1243 was increased from 2035 to 2407 and the metabolite number was increased from 1018 to 1094. Accordingly, the unique ORF number was increased from 1026 to 1243. To improve the metabolic functions of P. pastoris genome-scale metabolic model, the biosynthesis pathways of vitamins and cofactors were carefully added. iRY1243 showed good performances when predicting the growth capability on most of the reported carbon and nitrogen sources, the metabolic flux distribution with glucose as a sole carbon source, the essential and partially essential genes, and the effects of gene deletion or overexpression on cell growth and S-adenosyl-l-methionine production.
CONCLUSION: iRY1243 is an upgraded P. pastoris genome-scale metabolic model with significant improvements in the metabolic coverage and prediction ability, and thus it will be a potential platform for further systematic investigation of P. pastoris metabolism.
R. Aw, G. R. Barton, D. J. Leak, Appl Microbiol Biotechnol 101 5045 (2017).
Clonal variation, wherein a range of specific productivities of secreted proteins are observed from supposedly identical transformants, is an accepted aspect of working with Pichia pastoris. It means that a significant number of transformants need to be tested to obtain a representative sample, and in commercial protein production, companies regularly screen thousands of transformants to select for the highest secretor. Here, we have undertaken a detailed investigation of this phenomenon by characterising clones transformed with the human serum albumin gene. The titers of nine clones, each containing a single copy of the human serum albumin gene (identified by qPCR), were measured and the clones grouped into three categories, namely, high-, mid- and low-level secretors. Transcriptomic analysis, using microarrays, showed that no regulatory patterns consistently correlated with titer, suggesting that the causes of clonal variation are varied. However, a number of physiological changes appeared to underlie the differences in titer, suggesting there is more than one biochemical signature for a high-secreting strain. An anomalous low-secreting strain displaying high transcript levels that appeared to be nutritionally starved further emphasises the complicated nature of clonal variation.
Q. Zhou et al., J Biosci Bioeng (2017).
A novel thermostable endoglucanase (CTendo45) encoding gene was cloned from Chaetomium thermophilum and heterologously expressed in Pichia pastoris. Sequence alignment indicated that the CTendo45 enzyme belonged to glycoside hydrolase family 45. The recombinant enzyme was purified by Ni2+ affinity chromatography, and its apparent molecular mass was estimated to be 32 kDa by SDS-PAGE. The purified enzyme displayed maximum activity at 70 degrees C and pH 4. CTendo45 was stable at 60 degrees C for 1 h, and residual activities of 78.9% and 65.6% were estimated after 1 h at 70 degrees C and 80 degrees C, respectively. Ca2+, Zn2+, Mg2+, Cu2+ and Mn2+ were found to have beneficial effects on the enzyme activity to different degrees. The specific activity of purified CTendo45 was 1.52 IU mg-1 and the Km value was 59.6 mug ml-1 with a sodium carboxymethyl cellulose substrate. Moreover, CTendo45 exhibited high hydrolysis activity towards pectin, and the hydrolysis products were mainly galacturonic acid oligosaccharides. CTendo45 is the first reported bifunctional enzyme in glycoside hydrolase family 45 from C.thermophilum that is able to hydrolyze both cellulose and pectin. The biochemical properties of this recombinant CTendo45 make it a potentially effective glycoside hydrolase for industrial applications.
G. Agrawal, H. H. Shang, Z. J. Xia, S. Subramani, J Biol Chem (2017).
The peroxins Pex19 and Pex3 play an indispensable role in peroxisomal membrane protein (PMP) biogenesis, peroxisome division and inheritance. Pex19 plays multiple roles in these processes, but how these functions relate to the structural organization of the Pex19 domains is unresolved. To this end, using deletion mutants, we mapped the Pex19 regions required for peroxisome biogenesis in the yeast Pichia pastoris. Surprisingly, import-competent peroxisomes still formed when Pex19 domains previously believed to be required for biogenesis were deleted, although the peroxisome size was larger than that in wild-type cells. Moreover, these mutants exhibited a delay of 14-24 h in peroxisome biogenesis. The shortest functional N-terminal (NTCs) and C-terminal constructs (CTCs) were Pex19 (aa 1-150) and Pex19 (aa 89-300), respectively. Deletions of the N-terminal Pex3-binding site disrupted direct interactions of Pex19 with Pex3, but preserved interactions with a membrane peroxisomal targeting signal (mPTS)-binding PMP, Pex10. In contrast, deletion of the C-terminal mPTS-binding domain of Pex19 disrupted its interaction with Pex10, while leaving the Pex19-Pex3 interactions intact. However, Pex11 and Pex25 retained their interactions with both N- and C-terminal deletion mutants. NTC-CTC co-expression improved growth and reverted the larger-than-normal peroxisome size observed with the single deletions. Pex25 was critical for peroxisome formation with the CTC variants, and its overexpression enhanced their interactions with Pex3 and aided the growth of both NTC and CTC Pex19 variants. In conclusion, physical segregation of the Pex3 and PMP-binding domains of Pex19 has provided novel insights into the modular architecture of Pex19. We define the minimum region of Pex19 required for peroxisome biogenesis and a unique role for Pex25 in this process.
N. Adelantado et al., Microb Cell Fact 16 86 (2017).
BACKGROUND: Cultivation of recombinant Pichia pastoris (Komagataella sp.) under hypoxic conditions has a strong positive effect on specific productivity when the glycolytic GAP promoter is used for recombinant protein expression, mainly due to upregulation of glycolytic conditions. In addition, transcriptomic analyses of hypoxic P. pastoris pointed out important regulation of lipid metabolism and unfolded protein response (UPR). Notably, UPR that plays a role in the regulation of lipid metabolism, amino acid metabolism and protein secretion, was found to be upregulated under hypoxia.
RESULTS: To improve our understanding of the interplay between lipid metabolism, UPR and protein secretion, the lipidome of a P. pastoris strain producing an antibody fragment was studied under hypoxic conditions. Furthermore, lipid composition analyses were combined with previously available transcriptomic datasets to further understand the impact of hypoxia on lipid metabolism. Chemostat cultures operated under glucose-limiting conditions under normoxic and hypoxic conditions were analyzed in terms of intra/extracellular product distribution and lipid composition. Integrated analysis of lipidome and transcriptome datasets allowed us to demonstrate an important remodeling of the lipid metabolism under limited oxygen availability. Additionally, cells with reduced amounts of ergosterol through fluconazole treatment were also included in the study to observe the impact on protein secretion and its lipid composition.
CONCLUSIONS: Our results show that cells adjust their membrane composition in response to oxygen limitation mainly by changing their sterol and sphingolipid composition. Although fluconazole treatment results a different lipidome profile than hypoxia, both conditions result in higher recombinant protein secretion levels.
B. Wen et al., J Agric Food Chem 65 4676 (2017).
Currently, commercial plant peroxidases are all native and are isolated from plants such as horseradish and soybean. No recombinant plant peroxidase products have been available on the commercial market. The gene encoding peroxidase was cloned from windmill palm tree leaves. The codon-optimized gene was transformed into Pichia pastoris for expression. The recombinant windmill palm tree peroxidase (rWPTP) expressed by P. pastoris showed high stability under pH 2-10 and temperatures up to 70 degrees C to many metallic salts and organic solvents. The substrate specificity of WPTP was determined, and among the substrates tested, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was most suitable for WPTP. The Michaelis constants with the substrates H2O2 and ABTS were 4.6 x 10-4 and 1.6 x 10-4 M, respectively. The rWPTP expressed in P. pastoris may be a suitable enzyme for the biosynthesis of polymers because of its high stability and activity under acidic conditions.
S. Ben Azoun, H. Kallel, Microbiologyopen (2017).
Several factors affect protein expression in Pichia pastoris, one among them is the carbon source. In this work, we studied the effect of this factor on the expression level of rabies virus glycoprotein (RABV-G) in two recombinant clones harboring seven copies of the gene of interest. The expression was driven either by the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter or the inducible alcohol oxidase1 (AOX1) promoter. Clones were compared in terms of cell physiology and carbon source metabolism. The transcription levels of 16 key genes involved in the central metabolic pathway, the methanol catabolism, and the oxidative stress were investigated in both clones. Cell size, as a parameter reflecting cell physiological changes, was also monitored. Our results showed that when glucose was used as the sole carbon source, large cells were obtained. Transcript levels of the genes of the central metabolic pathway were also upregulated, whereas antioxidative gene transcript levels were low. By contrast, the use of methanol as a carbon source generated small cells and a shift in carbon metabolism toward the dissimilatory pathway by the upregulation of formaldehyde dehydrogenase gene and the downregulation of those of the central metabolic. These observations are in favor of the use of glucose to enhance the expression of RABV-G in P. pastoris.
A. Karnaouri et al., Biotechnol Biofuels 10 126 (2017).
BACKGROUND: Filamentous fungi are among the most powerful cellulolytic organisms in terrestrial ecosystems. To perform the degradation of lignocellulosic substrates, these microorganisms employ both hydrolytic and oxidative mechanisms that involve the secretion and synergism of a wide variety of enzymes. Interactions between these enzymes occur on the level of saccharification, i.e., the release of neutral and oxidized products, but sometimes also reflected in the substrate liquefaction. Although the synergism regarding the yield of neutral sugars has been extensively studied, further studies should focus on the oxidized sugars, as well as the effect of enzyme combinations on the viscosity properties of the substrates.
RESULTS: In the present study, the heterologous expression of an endoglucanase (EG) and its combined activity together with a lytic polysaccharide monooxygenase (LPMO), both from the thermophilic fungus Myceliophthora thermophila, are described. The EG gene, belonging to the glycoside hydrolase family 5, was functionally expressed in the methylotrophic yeast Pichia pastoris. The produced MtEG5A (75 kDa) featured remarkable thermal stability and showed high specific activity on microcrystalline cellulose compared to CMC, which is indicative of its processivity properties. The enzyme was capable of releasing high amounts of cellobiose from wheat straw, birch, and spruce biomass. Addition of MtLPMO9 together with MtEG5A showed enhanced enzymatic hydrolysis yields against regenerated amorphous cellulose (PASC) by improving the release not only of the neutral but also of the oxidized sugars. Assessment of activity of MtEG5A on the reduction of viscosity of PASC and pretreated wheat straw using dynamic viscosity measurements revealed that the enzyme is able to perform liquefaction of the model substrate and the natural lignocellulosic material, while when added together with MtLPMO9, no further synergistic effect was observed.
CONCLUSIONS: The endoglucanase MtEG5A from the thermophilic fungus M. thermophila exhibited excellent properties that render it a suitable candidate for use in biotechnological applications. Its strong synergism with LPMO was reflected in sugars release, but not in substrate viscosity reduction. Based on the level of oxidative sugar formation, this is the first indication of synergy between LPMO and EG reported.
X. Huang et al., J Med Virol (2017).
This study was designed to detect mutations that occur within the "a" determinant in the S gene of the hepatitis B virus (HBV) in patients with occult hepatitis B (OHB), and to analyze the influence of these mutations on expression and reactivity of the hepatitis B surface antigen (HBsAg). Twenty-three certified OHB samples were compared to 32 HBsAg positive samples from patients with chronic hepatitis B. The median HBV DNA levels in the OHB group were significantly lower than those in the control group (P < 0.0001). Mutations within the "a" determinant were analyzed by gene amplification and sequencing. This revealed mixed infections in which clones within a sample displayed either different mutations or mutations in association with clones that exhibited wild type amino acid patterns. Sequencing analysis also showed a significant difference between the proportions of amino acid mutations observed in the OHB and control groups. Seven recombinant S (rS) proteins with corresponding OHB mutations and three wild type alleles were expressed and purified in the Pichia pastoris expression system to preserve conformational attributes, and their reactivity analyzed using six commercial HBsAg assays. The OHB sera were HBsAg nonreactive while the rS proteins with corresponding OHB mutations were universally reactive. Thus, we postulate that the reduced binding affinity between mutated HBsAg and its antibody may not be as important in defining OHB as is the effect of specific mutations in the preS/S region of the genome that affect the synthesis and secretion of the S protein and/or the virion.
J. P. Schwarzhans et al., Front Microbiol 8 780 (2017).
Pichia pastoris is a non-conventional methylotrophic yeast that is widely used for recombinant protein production, typically by stably integrating the target gene into the genome as part of an expression cassette. However, the comparatively high clonal variability associated with this approach usually necessitates a time intense screening step in order to find strains with the desired productivity. Some of the factors causing this clonal variability can be overcome using episomal vectors containing an autonomously replicating sequence (ARS). Here, we report on the discovery, characterization, and application of a fragment of mitochondrial DNA from P. pastoris for use as an ARS. First encountered as an off-target event in an experiment aiming for genomic integration, the newly created circular plasmid named "pMito" consists of the expression cassette and a fragment of mitochondrial DNA. Multiple matches to known ARS consensus sequence motifs, but no exact match to known chromosomal ARS from P. pastoris were detected on the fragment, indicating the presence of a novel ARS element. Different variants of pMito were successfully used for transformation and their productivity characteristics were assayed. All analyzed clones displayed a highly uniform expression level, exceeding by up to fourfold that of a reference with a single copy integrated in its genome. Expressed GFP could be localized exclusively to the cytoplasm via super-resolution fluorescence microscopy, indicating that pMito is present in the nucleus. While expression levels were homogenous among pMito clones, an apparent upper limit of expression was visible that could not be explained based on the gene dosage. Further investigation is necessary to fully understand the bottle-neck hindering this and other ARS vectors in P. pastoris from reaching their full capability. Lastly, we could demonstrate that the mitochondrial ARS from P. pastoris is also suitable for episomal vector transformation in Saccharomyces cerevisiae, widening the potential for biotechnological application. pMito displayed strong potential to reduce clonal variability in experiments targeting recombinant protein production. These findings also showcase the as of yet largely untapped potential of mitochondrial ARS from different yeasts for biotechnological applications.
L. Galgoczy et al., Sci Rep 7 1963 (2017).
The recent global challenges to prevent and treat fungal infections strongly demand for the development of new antifungal strategies. The structurally very similar cysteine-rich antifungal proteins from ascomycetes provide a feasible basis for designing new antifungal molecules. The main structural elements responsible for folding, stability and antifungal activity are not fully understood, although this is an essential prerequisite for rational protein design. In this study, we used the Neosartorya fischeri antifungal protein (NFAP) to investigate the role of the disulphide bridges, the hydrophobic core, and the N-terminal amino acids in the formation of a highly stable, folded, and antifungal active protein. NFAP and its mutants carrying cysteine deletion (NFAPDeltaC), hydrophobic core deletion (NFAPDeltah), and N-terminal amino acids exchanges (NFAPDeltaN) were produced in Pichia pastoris. The recombinant NFAP showed the same features in structure, folding, stability and activity as the native protein. The data acquired with mass spectrometry, structural analyses and antifungal activity assays of NFAP and its mutants proved the importance of the disulphide bonding, the hydrophobic core and the correct N-terminus for folding, stability and full antifungal function. Our findings provide further support to the comprehensive understanding of the structure-function relationship in members of this protein group.
X. Q. Li et al., AMB Express 7 97 (2017).
To improve the temperature characteristics and catalytic efficiency of a glycoside hydrolase family (GHF) 11 xylanase from Aspergillus oryzae (AoXyn11A), its variants were predicted based on in silico design. Firstly, Gly21 with the maximum B-factor value, which was confirmed by molecular dynamics (MD) simulation on the three-dimensional structure of AoXyn11A, was subjected to site-saturation mutagenesis. Thus, one variant with the highest thermostability, AoXyn11AG21I, was selected from the mutagenesis library, E. coli/Aoxyn11A G21X (
X: any one of 20 amino acids). Secondly, based on the primary structure multiple alignment of AoXyn11A with seven thermophilic GHF11 xylanases, AoXyn11AY13F or AoXyn11AG21I-Y13F, was designed by replacing Tyr13 in AoXyn11A or AoXyn11AG21I with Phe. Finally, three variant-encoding genes, Aoxyn11A G21I, Aoxyn11A Y13F and Aoxyn11A G21I-Y13F, were constructed by two-stage whole-plasmid PCR method, and expressed in Pichia pastoris GS115, respectively. The temperature optimum (T opt) of recombinant (re) AoXyn11AG21I-Y13F was 60 degrees C, being 5 degrees C higher than that of reAoXyn11AG21I or reAoXyn11AY13F, and 10 degrees C higher than that of reAoXyn11A. The thermal inactivation half-life (t 1/2) of reAoXyn11AG21I-Y13F at 50 degrees C was 240 min, being 40-, 3.4- and 2.5-fold longer than those of reAoXyn11A, reAoXyn11AG21I and reAoXyn11AY13F. The melting temperature (T m) values of reAoXyn11A, reAoXyn11AG21I, reAoXyn11AY13F and reAoXyn11AG21I-Y13F were 52.3, 56.5, 58.6 and 61.3 degrees C, respectively. These findings indicated that the iterative mutagenesis of both Gly21Ile and Tyr13Phe improved the temperature characteristics of AoXyn11A in a synergistic mode. Besides those, the catalytic efficiency (k cat/K m) of reAoXyn11AG21I-Y13F was 473.1 mL mg-1 s-1, which was 1.65-fold higher than that of reAoXyn11A.
S. Zhang et al., Biotechnol Lett (2017).
OBJECTIVE: To engineer Pichia pastoris for heterologous production of cellulase from Musca domestica and explore its potential for industrial applications.
RESULTS: A new beta-glucosidase gene (bg), encoding 562 amino acids, was cloned from M. domestica by using rapid amplification of cDNA ends. The gene bg was linked to pPICZalphaA and expressed in P. pastoris with a yield of 500 mg l-1. The enzyme has the maximum activity with 27.6 U mg-1 towards cellulose. The beta-glucosidase has stable activity from 20 to 70 degrees C and can tolerate one-mole glucose. It has the maximum activities for salicin (25.9 +/- 1.8 U mg-1), cellobiose (40.1 +/- 2.3 U mg-1) and cellulose (27.6 +/- 3.5 U mg-1). The wide-range substrate activities of the beta-glucosidase were further verified by matrix-assisted laser desorption/ionization mass spectra. Structural analysis shows that the beta-glucosidase belongs to glycoside hydrolase family Iota and possesses O-glycosylation sites.
CONCLUSIONS: Thus, a multifunctional beta-glucosidase was expressed from M. domestica and provides a potential tool for industrial application of cellulose.
Y. Bartolo-Aguilar et al., AMB Express 7 95 (2017).
The production of recombinant biopharmaceutical proteins is a multi-billion dollar market. Protein recovery represents a major part of the production costs. Pichia pastoris is one of the microbial systems most used for the production of heterologous proteins. The use of a cold-induced promoter to express lytic enzymes in the yeast after the growth stage could reduce protein recovery costs. This study shows that a cold-shock can be applied to induce lysis of the yeast cells. A strain of P. pastoris was constructed in which the endogenous eng gene encoding a putative endo-beta-1,3-glucanase was overexpressed using the cold-shock induced promoter of the cctalpha gene from Saccharomyces cerevisiae. In the transgenic P. pastoris, the expression of eng increased 3.6-fold after chilling the cells from 30 to 4 degrees C (cold-shock stage) followed by incubation for 6 h (eng expression stage). The culture was heated to 30 degrees C for 6 h (ENG synthesis stage) and kept at 37 degrees C for 24 h (lysis stage). After this procedure the cell morphology changed, spheroplasts were obtained and cellular lysis was observed. Thus, a clone of P. pastoris was obtained, which undergoes autolysis after a cold-shock.
X. Qiu et al., Fish Shellfish Immunol 66 148 (2017).
Interleukin 26 (IL-26) gene has been identified in human, amphibian and teleost but not in rodents. It is well accepted that IL-26 was a crucial member of IL-10 family which acts as a pro-inflammatory cytokine in human. However, the role of IL-26 in regulating inflammation in lower vertebrates including teleost has not been defined yet. In the present study, grass carp IL-26 (gcIL-26) coding sequence was isolated and identified. Its chromosomal synteny was also analyzed, showing that gcIL-26 gene is flanked by IL-22 and IFN-gamma genes with the same transcriptional orientation as seen in human, amphibian and zebrafish. Given that zebrafish and grass carp IL-26 shared relatively low amino acid identities with human IL-26, the functional roles of fish IL-26 are indispensable to be elucidated. Accordingly, recombinant gcIL-26 (rgcIL-26) was prepared by using Pichia pastoris expression system, and it was found to be partially glycosylated. Using grass carp head kidney leucocytes as cell model, rgcIL-26 displayed the bioactivity to stimulate the mRNA expression of some pro-inflammatory cytokines including IL-8, IL-1beta and IL-6, while inhibit mRNA expression of an anti-inflammatory cytokine, IL-10. Moreover, rgcIL-26 also up-regulated inos expression and NO production in grass carp monocytes/macrophages, strengthening its pro-inflammatory properties in fish. Those results collectively demonstrated the functional role of IL-26 in regulating inflammatory response in fish.
X. Liu, M. Liang, Y. Liu, X. Fan, Microb Cell Fact 16 81 (2017).
BACKGROUND: Pyrethroids are potentially harmful to human health and ecosystems. It is necessary to develop some efficient strategies to degrade pyrethroid residues. Biodegradation is generally considered as a safe, efficient, and inexpensive way to eliminate environmental contaminants. To date, although several pyrethroid-hydrolyzing esterases have been cloned, there has been no report about a pyrethroid hydrolase with high hydrolytic activity, good stability, and high productivity, indispensable enzymatic properties in practical biodegradation. Almost all pyrethroid hydrolases are intracellular enzymes, which require complex extraction protocols and present issues in terms of easy inactivation and low production.
RESULTS: In this study, random mutagenesis was performed on one pyrethroid-hydrolyzing esterase, Sys410, to enhance its activity and thermostability. Two beneficial mutations, A171V and D256N, were obtained by random mutagenesis and gave rise to the mutant M2. The mutant displayed ~1.5-fold improvement in the kcat/Km value and 2.46-fold higher catalytic activity. The optimal temperature was 10 degrees C higher than that of the wild-type enzyme (55 degrees C). The half-life at 40-65 degrees C was 3.3-310 times longer. It was surprising that M2 has a half-life of 12 h at 70 degrees C while Sys410 was completely inactivated at 70 degrees C. In addition, the desired gene was extracellularly expressed in a Pichia pastoris host system. The soluble expression level reached up to 689.7 mg/L. Remarkably, the enzyme could efficiently degrade various pyrethroids at moderate temperature for 15 min, exceeding a hydrolysis rate of 98%, which is the highest value ever reported.
CONCLUSIONS: This is the first report about random mutagenesis and secretory expression of pyrethroid-hydrolyzing esterase with high-level productivity and purity in P. pastoris. Broad substrate specificity, enhanced activity and thermostability make M2 an ideal candidate for the biodegradation of pyrethroid residues.
W. Yang et al., Sci Rep 7 1587 (2017).
As a feed additive, xylanase has been widely applied in the feed of monogastric animals, which contains multiple plant polysaccharides. However, during feed manufacture, the high pelleting temperatures challenge wild-type xylanases. The aim of this study was to improve the thermostability of Aspergillus sulphureus acidic xylanase. According to the predicted protein structure, a series of disulphide bridges and proline substitutions were created in the xylanase by PCR, and the mutants were expressed in Pichia pastoris. Enzyme properties were evaluated following chromatographic purification. All the recombinant enzymes showed optima at pH 3.0 and 50 degrees C or 55 degrees C and better resistance to some chemicals except for CuSO4. The specific activity of the xylanase was decreased by introduction of the mutations. Compared to the wild-type enzyme, a combined mutant, T53C-T142C/T46P, with a disulphide bond at 53-142 and a proline substitution at 46, showed a 22-fold increase of half-life at 60 degrees C. In a 10-L fermentor, the maximal xylanase activity of T53C-T142C/T46P reached 1,684 U/mL. It was suggested that the T53C-T142C/T46P mutant xylanase had excellent thermostability characteristics and could be a prospective additive in feed manufacture.
B. Ranjan, S. Pillai, K. Permaul, S. Singh, Bioresour Technol 238 582 (2017).
A recombinant Pichia pastoris harbouring the cyanate hydratase gene (rTl-Cyn) from the thermophilic fungus Thermomyces lanuginosus SSBP yielded a high titre of extracellular cyanate hydratase (100+/-13UmL-1) which was approximately 10-fold higher than the native fungal strain. The purified rTl-Cyn had a molecular mass of approximately 20kDa on SDS-PAGE, with Km, Vmax, kcat and kcat/Km values of 0.34mM, 2857.14micromolesmg-1min-1, 2.14x104s-1 and 6.3 x107M-1s-1, respectively. Its properties of thermostability, pH stability, and heavy metals insensitivity, make it a suitable candidate for bioremediation in extreme environments. The rTl-Cyn was able to degrade toxic cyanate completely with the liberation of ammonia, which was confirmed by FTIR analysis. This is the first report of any known cyanate hydratase that has been expressed in P. pastoris, characterized and effectively evaluated for cyanate detoxification.
O. V. Berezina et al., Appl Microbiol Biotechnol (2017).
A xyloglucanase of the GH74 family was identified in the thermophilic fungus strain Myceliophthora thermophila VKPM F-244, and its gene sequence was optimized for cloning and expression in Pichia pastoris. The recombinant xyloglucanase MtXgh74 exhibited the highest activity toward tamarind seed xyloglucan with a K M value of 0.51 +/- 0.06 mg/mL. The activities on barley beta-glucan and carboxymethylcellulose were about 4 and 2%, respectively, compared to xyloglucan. Maximum xyloglucanase activity was observed at 70-75 degrees C and pH 6.5. After pre-incubation at 50 degrees C, pH 6.0 for 3 h, the enzyme retained 100% of its activity. The half-life of MtXgh74 at 60 degrees C, pH 6.0 was 40 min. In P. pastoris, MtXgh74 was produced in glycosylated form. The enzyme production in a 1 L bioreactor resulted in a yield of 118 U/mL or 5.3 g/L after 51 h fermentation. Kinetic studies of the hydrolysis product formation suggest that MtXgh74 has an endo-processive mode of action. The final products were the standard xyloglucan building blocks XXXG, XXLG, XLXG, and XLLG. Additionally, MtXgh74 hydrolyzed various linkages within the xyloglucan building blocks XXXG, XXLG, and XLXG (except XLLG) producing diverse low molecular weight oligosaccharides which may be identified by MALDI-TOF as XG, XX, XXG/GXX/XGX, XXX, LG, LX/XL, XLX/XXL, LLG, GXXXG, GXLLG, XLLGX. The unique combination of different activities within one enzyme along with its high thermostability and specificity toward xyloglucan makes MtXgh74 a promising candidate enzyme for industrial applications.
E. Rampler et al., Analyst 142 1891 (2017).
Quantification is an essential task in comprehensive lipidomics studies challenged by the high number of lipids, their chemical diversity and their dynamic range of the lipidome. In this work, we introduce lipidome isotope labeling of yeast (LILY) in order to produce (non-radioactive) isotopically labeled eukaryotic lipid standards in yeast for normalization and quantification in mass spectrometric assays. More specifically, LILY is a fast and efficient in vivo labeling strategy in Pichia pastoris for the production of 13C labeled lipid library further paving the way to comprehensive compound-specific internal standardization in quantitative mass spectrometry based assays. More than 200 lipid species (from PA, PC, PE, PG, PI, PS, LysoGP, CL, DAG, TAG, DMPE, Cer, HexCer, IPC, MIPC) were obtained from yeast extracts with an excellent 13C enrichment >99.5%, as determined by complementary high resolution mass spectrometry based shotgun and high resolution LC-MS/MS analysis. In a first proof of principle study we tested the relative and absolute quantification capabilities of the 13C enriched lipids obtained by LILY using a parallel reaction monitoring based LC-MS approach. In relative quantification it could be shown that compound specific internal standardization was essential for the accuracy extending the linear dynamic range to four orders of magnitude. Excellent analytical figures of merit were observed for absolute quantification for a selected panel of 5 investigated glycerophospholipids (e.g. LOQs around 5 fmol absolute; typical concentrations ranging between 1 to 10 nmol per 108 yeast cell starting material; RSDs <10% (N = 4)).
A. K. Maciola et al., Front Immunol 8 444 (2017).
Hemagglutinin glycoprotein (HA) is a principle influenza vaccine antigen. Recombinant HA-based vaccines become a potential alternative for traditional approach. Complexity and variation of HA N-glycosylation are considered as the important factors for the vaccine design. The number and location of glycan moieties in the HA molecule are also crucial. Therefore, we decided to study the effect of N-glycosylation pattern on the H5 antigen structure and its ability to induce immunological response. We also decided to change neither the number nor the position of the HA glycosylation sites but only the glycan length. Two variants of the H5 antigen with high mannose glycosylation (H5hm) and with low-mannose glycosylation (H5Man5) were prepared utilizing different Pichia strains. Our structural studies demonstrated that only the highly glycosylated H5 antigen formed high molecular weight oligomers similar to viral particles. Further, the H5hm was much more immunogenic for mice than H5Man5. In summary, our results suggest that high mannose glycosylation of vaccine antigen is superior to the low glycosylation pattern. Our findings have strong implications for the recombinant HA-based influenza vaccine design.
S. L. Martinez-Hernandez et al., Biotechnol Lett (2017).
OBJECTIVE: To generate an immunogenic chimeric protein containing the Entamoeba histolytica LC3 fragment fused to the retrograde delivery domains of exotoxin A of Pseudomonas aeruginosa and KDEL3 for use as an effective vaccine.
RESULTS: A codon-optimized synthetic gene encoding the PEDeltaIII-LC3-KDEL3 fusion construct was designed for expression in Pichia pastoris. This transgene was subcloned into the plasmid pPIC9 for methanol-inducible expression. After transformation and selection of positive-transformed clones by PCR, the expression of the recombinant protein PEDeltaIII-LC3-KDEL3 was elicited. SDS-PAGE, protein glycosylation staining and western blot assays demonstrated a 67 kDa protein in the medium culture supernatant. The recombinant protein was detected with a polyclonal anti-6X His tag antibody and a polyclonal E. histolytica-specific antibody. A specific antibody response was induced in hamsters after immunization with this protein.
CONCLUSIONS: We report for the first time the design and expression of the recombinant E. histolytica LC3 protein fused to PEDeltaIII and KDEL3, with potential application as an immunogen.
D. P. Luiz et al., Microb Cell Fact 16 76 (2017).
BACKGROUND: Antimicrobial peptides (AMPs) are the first line of host immune defense against pathogens. Among AMPs from the honeybee Apis mellifera, abaecin is a major broad-spectrum antibacterial proline-enriched cationic peptide.
RESULTS: For heterologous expression of abaecin in Pichia pastoris, we designed an ORF with HisTag, and the codon usage was optimized. The gene was chemically synthetized and cloned in the pUC57 vector. The new ORF was sub-cloned in the pPIC9 expression vector and transformed into P. pastoris. After selection of positive clones, the expression was induced by methanol. The supernatant was analyzed at different times to determine the optimal time for the recombinant peptide expression. As a proof-of-concept, Escherichia coli was co-incubated with the recombinant peptide to verify its antimicrobial potential.
DISCUSSION: Briefly, the recombinant Abaecin (rAbaecin) has efficiently decreased E. coli growth (P < 0.05) through an in vitro assay, and may be considered as a novel therapeutic agent that may complement other conventional antibiotic therapies.
M. H. Liang, S. S. Zhou, J. G. Jiang, Enzyme Microb Technol 102 74 (2017).
HBscFv-IFNgamma, a fusion protein constructed by fusing gamma-interferon (IFNgamma) with an antibody fragment HBscFv for the purpose of targeted delivery of the cytokine IFNgamma, was designed in order to enhance its therapeutic efficacy through increasing its hepatoma localization. HBscFv and IFNgamma were connected into HBscFv-IFNgamma by the linker (Gly4Ser)3, and then the multicopy recombinant plasmids pPICZalphaA/(HBscFv-IFNgamma)1,2,4 were constructed and transformed into Komagatella (Pichia) pastoris X33. The engineering strain X4, which had much higher copy number and could secretively express HBscFv-IFNgamma, was screened from transformed X33 by qPCR. Results from SDS-PAGE, Western blotting and ELISA indicated that HBscFv-IFNgamma displayed an excellent immunoreaction against HBsAg. The culture supernatant of X4 was purified by 14F7 affinity chromatography to obtain the fusion protein HBscFv-IFNgamma in a purity of 95-98%. The HBscFv-IFNgamma was able to bind 27.9% HBsAg in the serum of HBV transgenic mice, showing that the antibody of HBscFv-IFNgamma has high binding affinity against HBsAg. The expressing of the recombinant HBscFv-IFNgamma in P. pastoris provides a promising and inexpensive diagnostic reagent for preventing HBV infection.
X. G. Zhang et al., Protein Pept Lett (2017).
Delta sleep-inducing peptide (DSIP) is a nonapeptide that could promote sleep through the induction of slow wave sleep. To further study the pharmacological effect of DSIP on insomnia, we designed a fusion protein containing N-terminal TAT-based transduction domain followed by human serum albumin and DSIP and designated this protein as PHD fusion protein. We expressed PHD fusion protein in Pichia pastoris, purified it to near homogeneity by three-step chromatography and performed bioactivity assay. The pharmacological activity of PHD fusion protein was studied using classic pentobarbital-induced sleep test in healthy mice. PHD fusion protein increased the hypnotic effects of pentobarbital by reducing sleep latency and prolonged sleep duration. The present study suggested PHD fusion protein could be a new drug candidate for insomnia.
W. H. Chen et al., J Pharm Sci (2017).
From 2002 to 2003, a global pandemic of severe acute respiratory syndrome (SARS) spread to 5 continents and caused 8000 respiratory infections and 800 deaths. To ameliorate the effects of future outbreaks as well as to prepare for biodefense, a process for the production of a recombinant protein vaccine candidate is under development. Previously, we reported the 5 L scale expression and purification of a promising recombinant SARS vaccine candidate, RBD219-N1, the 218-amino acid residue receptor-binding domain (RBD) of SARS coronavirus expressed in yeast-Pichia pastoris X-33. When adjuvanted with aluminum hydroxide, this protein elicited high neutralizing antibody titers and high RBD-specific antibody titers. However, the yield of RBD219-N1 (60 mg RBD219-N1 per liter of fermentation supernatant; 60 mg/L FS) still required improvement to reach our target of >100 mg/L FS. In this study, we optimized the 10 L scale production process and increased the fermentation yield 6- to 7-fold to 400 mg/L FS with purification recovery >50%. A panel of characterization tests indicated that the process is reproducible and that the purified, tag-free RBD219-N1 protein has high purity and a well-defined structure and is therefore a suitable candidate for production under current Good Manufacturing Practice and future phase-1 clinical trials.
M. Wang et al., Plant Cell Rep 36 1125 (2017).
KEY MESSAGE: CsHSP17.7, CsHSP18.1, and CsHSP21.8 expressions are induced by heat and cold stresses, and CsHSP overexpression confers tolerance to heat and cold stresses in transgenic Pichia pastoris and Arabidopsis thaliana. Small heat shock proteins (sHSPs) are crucial for protecting plants against biotic and abiotic stresses, especially heat stress. However, knowledge concerning the functions of Camellia sinensis sHSP in heat and cold stresses remains poorly understood. In this study, three C. sinensis sHSP genes (i.e., CsHSP17.7, CsHSP18.1, and CsHSP21.8) were isolated and characterized using suppression subtractive hybridization (SSH) technology. The CsHSPs expression levels in C. sinensis leaves were significantly up-regulated by heat and cold stresses. Phylogenetic analyses revealed that CsHSP17.7, CsHSP18.1, and CsHSP21.8 belong to sHSP Classes I, II, and IV, respectively. Heterologous expression of the three CsHSP genes in Pichia pastoris cells enhanced heat and cold stress tolerance. When exposed to heat and cold treatments, transgenic Arabidopsis thaliana plants overexpressing CsHSP17.7, CsHSP18.1, and CsHSP21.8 had lower malondialdehyde contents, ion leakage, higher proline contents, and transcript levels of stress-related genes (e.g., AtPOD, AtAPX1, AtP5CS2, and AtProT1) compared with the control line. In addition, improved seed germination vigor was also observed in the CsHSP-overexpressing seeds under heat stress. Taken together, our results suggest that the three identified CsHSP genes play key roles in heat and cold tolerance.
T. Huyan et al., Oncol Lett 13 1539 (2017).
Human aspartyl-(asparaginyl)-beta-hydroxylase (HAAH) has recently been the subject of several studies, as it was previously observed to be overexpressed in numerous types of carcinoma cells and tissues in patient tumor samples. HAAH has been implicated in tumor invasion and metastasis, indicating that it may be an important target and biomarker for tumor diagnosis and treatment. However, the immunological tools currently available for the study of this protein, including monoclonal antibodies, are limited, as is the present knowledge regarding the role of HAAH in tumor therapy and diagnosis. In the present study, a recombinant C-terminal domain of HAAH was expressed in Pichia pastoris and a novel monoclonal antibody (mAb) targeting HAAH (HAAH-C) was constructed. Immunofluorescence and antibody-dependent cellular cytotoxicity (ADCC) assays were used to demonstrate the specificity and ADCC activity of this antibody. The results demonstrated that this anti-C-terminal HAAH mAB, in combination with an existing anti-N terminal HAAH mAb, exhibited a high response to native HAAH from carcinoma cell culture supernatant, as measured with a double antibody sandwich enzyme-linked immunosorbent assay. This validated novel mAB-HAAH-C may prompt further studies into the underlying mechanisms of HAAH, and the exploration of its potential in tumor diagnosis and therapy.
M. Wang et al., Sci Rep 7 1237 (2017).
Small heat shock proteins (sHSPs) play important roles in responses to heat stress. However, the functions of sHSPs in tea plants (Camellia sinensis) remain uncharacterized. A novel sHSP gene, designated CsHSP17.2, was isolated from tea plants. Subcellular localization analyses indicated that the CsHSP17.2 protein was present in the cytosol and the nucleus. CsHSP17.2 expression was significantly up-regulated by heat stress but was unaffected by low temperature. The CsHSP17.2 transcript levels increased following salt and polyethylene glycol 6000 treatments but decreased in the presence of abscisic acid. The molecular chaperone activity of CsHSP17.2 was demonstrated in vitro. Transgenic Escherichia coli and Pichia pastoris expressing CsHSP17.2 exhibited enhanced thermotolerance. The transgenic Arabidopsis thaliana exhibited higher maximum photochemical efficiencies, greater soluble protein proline contents, higher germination rates and higher hypocotyl elongation length than the wild-type controls. The expression levels of several HS-responsive genes increased in transgenic A. thaliana plants. Additionally, the CsHSP17.2 promoter is highly responsive to high-temperature stress in A. thaliana. Our results suggest that CsHSP17.2 may act as a molecular chaperone to mediate heat tolerance by maintaining maximum photochemical efficiency and protein synthesis, enhancing the scavenging of reactive oxygen species and inducing the expression of HS-responsive genes.
K. Noridomi et al., Elife 6 (2017).
The nicotinic acetylcholine receptor (nAChR) is a major target of autoantibodies in myasthenia gravis (MG), an autoimmune disease that causes neuromuscular transmission dysfunction. Despite decades of research, the molecular mechanisms underlying MG have not been fully elucidated. Here, we present the crystal structure of the nAChR alpha1 subunit bound by the Fab fragment of mAb35, a reference monoclonal antibody that causes experimental MG and competes with ~65% of antibodies from MG patients. Our structures reveal for the first time the detailed molecular interactions between MG antibodies and a core region on nAChR alpha1. These structures suggest a major nAChR-binding mechanism shared by a large number of MG antibodies and the possibility to treat MG by blocking this binding mechanism. Structure-based modeling also provides insights into antibody-mediated nAChR cross-linking known to cause receptor degradation. Our studies establish a structural basis for further mechanistic studies and therapeutic development of MG.
R. Wang et al., J Steroid Biochem Mol Biol 171 254 (2017).
Steroid 11-hydroxylation by filamentous fungi is a major route for industrial scale production of key intermediates in the synthesis of steroid drugs. Although it is well established that enzymes involved in fungal hydroxylation of steroids are cytochrome P450s (CYP), few fungal steroid hydroxylase genes have been identified. In this study, we identified a novel 11alpha-hydroxylase gene CYP5311B1 from Absidia coerulea AS3.65 by a combination of transcriptome sequencing, real-time qRT-PCR and heterologous expression in Pichia pastoris. The full-length open reading frame (ORF) of CYP5311B1 is predicted to encode a CYP protein of 527 amino acids whose expression in Pichia cells was confirmed by western blot. In addition, the major hydroxylation product was characterized by HPLC and 2D NMR. CYP5311B1 was highly induced by steroid substrate at the transcriptional level. The cloning and identification of an 11alpha-hydroxylase gene from A. coerulea should aid in a better understanding of the structural basis underlying regio- and stereoselectivity, and substrate specificity of fungal steroid 11alpha-hydroxylases, thus facilitating the engineering of more efficient steroid hydroxylases for industrial applications.
S. Pourasadi, S. L. Mousavi Gargari, M. Rajabibazl, S. Nazarian, Turk J Med Sci 47 695 (2017).
AIM: Helicobacter pylori is a major health problem. One of the therapeutic approaches is administration of antibody against H. pylori. The methylotrophic Pichia pastoris is a suitable host for expression of recombinant antibody fragments. The aims of this study were the expression and the evaluation of camelid nanobody in the yeast Pichia pastoris.
MATERIALS AND METHODS: The camelid-derived heavy-chain antibody (nanobody) against the UreC subunit of urease from H. pylori was subcloned in the pPink-HC shuttle vector and transferred into Escherichia coli TOP10. After digestion and purification, the shuttle vector was transformed in the PichiaPink expression system. The expression was evaluated in an in vitro system.
RESULTS: The yield of the nanobody expressed in P. pastoris was estimated to be 5 mg/L as compared to 2 mg/L expressed by E. coli. The nanobody was purified and binding affinity to the UreC antigen was evaluated using ELISA. Neutralization abilities of the two nanobodies expressed in yeast and E. coli were compared. The yeast-expressed nanobody specifically detected recombinant UreC and inhibited urease activity with high efficiency.
CONCLUSION: The results suggest attribution of the enhanced quality and quantity of the nanobody produced in P. pastoris to better posttranslational modification and folding in the yeast cell.
Y. Cai et al., Oncotarget 8 20187 (2017).
The over-expression of basic fibroblast growth factor (bFGF) plays a crucial role in the development, invasion and metastasis of lung cancer. Therefore, neutralizing antibodies against bFGF may inhibit the growth of lung cancer. In this study, a Disulfide-stabilized diabody (ds-Diabody) against bFGF was constructed by site-directed mutation and overlap extension PCR (SOE-PCR) at the position of VH44 and VL100 in the scFv. The ds-Diabody was constructed and expressed in Pichia pastoris. We found that the ds-Diabody against bFGF could efficiently suppress the proliferation, migration and invasion of human lung cancer A549 cells in vitro. Moreover, in A549 cells, the ds-Diabody against bFGF could inhibit bFGF-induced activation of downstream signaling regulators, such as phospho-Akt and phospho-MAPK. In the nude mouse xenograft model of lung cancer, the ds-Diabody against bFGF could significantly inhibit tumor growth and decrease the densities of micro-vessels and lymphatic vessels in tumor tissue. Our data indicate that the ds-Diabody against bFGF could effectively suppress the lung cancer growth through blockade of bFGF signaling pathway and inhibition of tumor angiogenesis, which may make it a potential therapeutic candidate antibody drug for human lung cancer therapy.
E. D. Morales-Alvarez et al., Enzyme Res 2017 5947581 (2017).
Laccases are multicopper oxidases that catalyze aromatic and nonaromatic compounds with concomitant reduction of molecular oxygen to water. They are of great interest due to their potential biotechnological applications. In this work we statistically improved culture media for recombinant GILCC1 (rGILCC1) laccase production at low scale from Ganoderma lucidum containing the construct pGAPZalphaA-GlucPost-Stop in Pichia pastoris. Temperature, pH stability, and kinetic parameter characterizations were determined by monitoring concentrate enzyme oxidation at different ABTS substrate concentrations. Plackett-Burman Design allowed improving enzyme activity from previous work 36.08-fold, with a laccase activity of 4.69 +/- 0.39 UL-1 at 168 h of culture in a 500 mL shake-flask. Concentrated rGILCC1 remained stable between 10 and 50 degrees C and retained a residual enzymatic activity greater than 70% at 60 degrees C and 50% at 70 degrees C. In regard to pH stability, concentrated enzyme was more stable at pH 4.0 +/- 0.2 with a residual activity greater than 90%. The lowest residual activity greater than 55% was obtained at pH 10.0 +/- 0.2. Furthermore, calculated apparent enzyme kinetic parameters were a Vmax of 6.87 x 10-5 mM s-1, with an apparent Km of 5.36 x 10-2 mM. Collectively, these important stability findings open possibilities for applications involving a wide pH and temperature ranges.
G. Resendiz-Cardiel, R. Arroyo, J. Ortega-Lopez, Protein Expr Purif 134 104 (2017).
The legumain-like cysteine proteinase TvLEGU-1 from Trichomonas vaginalis plays a major role in trichomonal cytoadherence. However, its structure-function characterization has been limited by the lack of a reliable recombinant expression platform to produce this protein in its native folded conformation. TvLEGU-1 has been expressed in Escherichia coli as inclusion bodies and all efforts to refold it have failed. Here, we describe the expression of the synthetic codon-optimized tvlegu-1 (tvlegu-1-opt) gene in Pichia pastoris strain X-33 (Mut+) under the inducible AOX1 promoter. The active TvLEGU-1 recombinant protein (rTvLEGU-1) was secreted into the medium when tvlegu-1-opt was fused to the Aspergillus niger alpha-amylase signal peptide. The rTvLEGU-1 secretion was influenced by the gene copy number and induction temperature. Data indicate that increasing tvlegu-1-opt gene copy number was detrimental for heterologous expression of the enzymatically active TvLEGU-1. Indeed, expression of TvLEGU-1 had a greater impact on cell viability for those clones with 26 or 29 gene copy number, and cell lysis was observed when the induction was carried out at 30 degrees C. The enzyme activity in the medium was higher when the induction was carried out at 16 degrees C and in P. pastoris clones with lower gene copy number. The results presented here suggest that both copy number and induction temperature affect the rTvLEGU-1 expression in its native-like and active conformation.
T. Hiragun et al., J Dermatol Sci 87 3 (2017).
Sweat is an exacerbation factor in atopic dermatitis (AD) in all age groups. A body core temperature elevation with sweating triggers cholinergic urticaria (CholU). We recently reported that AD symptoms are improved by tannic acid-containing spray, which suppresses the basophil histamine release induced by semi-purified sweat antigen in vitro, and by showering, which removes antigens in sweat from the skin surface. Sweat contains small amount of proteins including proteases, protease inhibitors, and anti-microbial peptides. We finally identified MGL_1304 secreted by Malassezia (M.) globosa as a major histamine - releasing antigen in human sweat. MGL_1304 is a 17-kDa protein in sweat that elicits almost the highest histamine - release activity from basophils of patients with AD and CholU among antigens derived from Malassezia species. Moreover, serum levels of anti-MGL_1304 IgE were significantly higher in patients with AD and CholU than in normal controls. The recombinant protein produced by Pichia pastoris possessed comparable allergenicity to native MGL_1304. We found a monoclonal IgE antibody against MGL_1304 which did not elicit histamine release from sensitized mast cells. Desensitization therapy using autologous sweat, or MGL_1304 purified from culture of M. globosa or its cognates might be beneficial for patients with intractable CholU due to sweat allergy.
H. Ehara, T. Umehara, S. I. Sekine, S. Yokoyama, Biochem Biophys Res Commun 487 230 (2017).
RNA polymerase II (Pol II) is a 12-subunit protein complex that conducts the transcription of mRNA and some small RNAs. In this work, the crystal structure of Pol II from the methylotropic yeast Komagataella pastoris (Pichia pastoris) was determined. While the structure is highly homologous to that of Pol II from the budding yeast Saccharomyces cerevisiae, the stalk and clamp modules of the K. pastoris Pol II displayed large inward rotations, closing the central cleft to a greater extent than in the known S. cerevisiae Pol II structures. The conformational differences reflect the inherent flexibilities of the stalk and the clamp, as additional low-resolution structures of K. pastoris Pol II in different crystal forms revealed diverse stalk and clamp orientations. Comparisons with other eukaryotic/archaeal RNA polymerase structures in the Protein Data Bank revealed the distributions of the stalk and clamp orientations. The conformational plasticity should be essential for transcriptional functions and binding various regulatory factors.
K. B. Rodrigues et al., Carbohydr Res (2017).
The discovery of lytic polysaccharides monooxygenases copper dependent (LPMOs) revolutionized the classical concept that the cleavage of cellulose is a hydrolytic process in recent years. These enzymes carry out oxidative cleavage of cellulose (and other polysaccharides), acting synergistically with cellulases and other hydrolases. In fact, LPMOs have the potential for increasing the efficiency of the lignocellulosic biomass conversion in biofuels and high value chemicals. Among a small number of microbial LPMOs that have been characterized, some LPMOs were expressed and characterized biochemically from the bacteria Thermobifida fusca, using the host Escherichia coli. In this work, the E7 LPMO protein of T. fusca was expressed both in E. coli (native DNA sequence) and Pichia pastoris (codon-optimized DNA sequence), for further analysis of oxidative cleavage, with PASC (phosphoric acid swollen cellulose) and Avicel PH-101 substrates, using mass spectrometry analysis. Mass spectra results of Avicel PH-101 and PASC cleavages by purified E7 LPMO expressed in E. coli and in P. pastoris allowed the visualization of compounds corresponding to oxidized and non-oxidized oligosaccharides. Further optimization of reactions will be performed, since it was found only one degree of polymerization (DP 7). This work demonstrated that it is possible to produce the E7 LPMO from T. fusca in the host P. pastoris, and the recombinant protein was shown to be active on cellulose. The approach used in the present work could be an alternative to produce this bacterial LPMO for cellulose cleavage.
Y. T. Chang et al., PLoS One 12 e0175321 (2017).
Rhodostomin (Rho) is a medium disintegrin containing a 48PRGDMP motif. We here showed that Rho proteins with P48A, M52W, and P53N mutations can selectively inhibit integrin alphaIIbbeta3. To study the roles of the RGD loop and C-terminal region in disintegrins, we expressed Rho 48PRGDMP and 48ARGDWN mutants in Pichia pastoris containing 65P, 65PR, 65PRYH, 65PRNGLYG, and 65PRNPWNG C-terminal sequences. The effect of C-terminal region on their integrin binding affinities was alphaIIbbeta3 > alphavbeta3 >/= alpha5beta1, and the 48ARGDWN-65PRNPWNG protein was the most selective integrin alphaIIbbeta3 mutant. The 48ARGDWN-65PRYH, 48ARGDWN-65PRNGLYG, and 48ARGDWN-65PRNPWNG mutants had similar activities in inhibiting platelet aggregation and the binding of fibrinogen to platelet. In contrast, 48ARGDWN-65PRYH and 48ARGDWN-65PRNGLYG exhibited 2.9- and 3.0-fold decreases in inhibiting cell adhesion in comparison with that of 48ARGDWN-65PRNPWNG. Based on the results of cell adhesion, platelet aggregation and the binding of fibrinogen to platelet inhibited by ARGDWN mutants, integrin alphaIIbbeta3 bound differently to immobilized and soluble fibrinogen. NMR structural analyses of 48ARGDWN-65PRYH, 48ARGDWN-65PRNGLYG, and 48ARGDWN-65PRNPWNG mutants demonstrated that their C-terminal regions interacted with the RGD loop. In particular, the W52 sidechain of 48ARGDWN interacted with H68 of 65PRYH, L69 of 65PRNGLYG, and N70 of 65PRNPWNG, respectively. The docking of the 48ARGDWN-65PRNPWNG mutant into integrin alphaIIbbeta3 showed that the N70 residue formed hydrogen bonds with the alphaIIb D159 residue, and the W69 residue formed cation-pi interaction with the beta3 K125 residue. These results provide the first structural evidence that the interactions between the RGD loop and C-terminus of medium disintegrins depend on their amino acid sequences, resulting in their functional differences in the binding and selectivity of integrins.
T. Mairinger, S. Hann, Anal Bioanal Chem 409 3713 (2017).
A novel analytical approach based on liquid chromatography coupled to quadrupole time of flight mass spectrometry, employing data-dependent triggering for analysis of isotopologue and tandem mass isotopomer fractions of metabolites of the primary carbon metabolism was developed. The implemented QTOFMS method employs automated MS/MS triggering of higher abundant, biologically relevant isotopologues for generating positional information of the respective metabolite. Using this advanced isotopologue selective fragmentation approach enables the generation of significant tandem mass isotopomer data within a short cycle time without compromising sensitivity. Due to a lack of suitable reference material certified for isotopologue ratios, a Pichia pastoris cell extract with a defined 13C distribution as well as a cell extract from a 13C-based metabolic flux experiment were employed for proof of concept. Moreover, a method inter-comparison with an already established GC-CI-(Q)TOFMS approach was conducted. Both methods showed good agreement on isotopologue and tandem mass isotopomer distributions for the two different cell extracts. Graphical abstract Schematic overview of data-dependent isotopologue fragmentation for acquisition of isotopologue and tandem mass isotopomer fractions.
L. Damasceno, G. Ritter, C. A. Batt, Protein Expr Purif 134 72 (2017).
The trematode Schistosoma mansoni Sm14 antigen was expressed in the yeast Pichia pastoris and secreted into the culture medium at yields of approximately 250 mg L-1. Sm14 belongs to a family of fatty-acid binding proteins and appears to play an important role in uptake, transport, and compartmentalization of lipids in S. mansoni and it is a potential vaccine candidate in both humans and domesticated animals. The Sm14 gene was codon-optimized for expression in P. pastoris, and placed under transcription of the strong methanol inducible AOX1 promoter. Mut+ transformants were selected and used in fed-batch cultivation using a 2.5L fermenter equipped with an on-line methanol control system in order to maintain constant methanol levels during induction. Optimal conditions for the expression of Sm14 by P. pastoris were found to be: dissolved oxygen at 40%, temperature of 25 degrees C, pH 5.0, and a constant methanol concentration of 1 gL-1. Our results show that a correctly processed Sm14 was secreted into the culture medium at levels of approximately 250 mg L-1. Sm14 from clarified culture medium was purified using a two-step procedure: anion-exchange chromatography followed by hydrophobic interaction chromatography, resulting in >95% purity with a final yield of 40% from the starting cell culture medium. This product has been tested in preliminary clinical trials and shown to elicit an antibody response with no adverse reactions.
B. Franco-Orozco et al., New Phytol 214 1657 (2017).
Pathogen-associated molecular patterns (PAMPs) are detected by plant pattern recognition receptors (PRRs), which gives rise to PAMP-triggered immunity (PTI). We characterized a novel fungal PAMP, Cell Death Inducing 1 (RcCDI1), identified in the Rhynchosporium commune transcriptome sampled at an early stage of barley (Hordeum vulgare) infection. The ability of RcCDI1 and its homologues from different fungal species to induce cell death in Nicotiana benthamiana was tested following agroinfiltration or infiltration of recombinant proteins produced by Pichia pastoris. Virus-induced gene silencing (VIGS) and transient expression of Phytophthora infestans effectors PiAVR3a and PexRD2 were used to assess the involvement of known components of PTI in N. benthamiana responses to RcCDI1. RcCDI1 was highly upregulated early during barley colonization with R. commune. RcCDI1 and its homologues from different fungal species, including Zymoseptoria tritici, Magnaporthe oryzae and Neurospora crassa, exhibited PAMP activity, inducing cell death in Solanaceae but not in other families of dicots or monocots. RcCDI1-triggered cell death was shown to require N. benthamiana Brassinosteroid insensitive 1-Associated Kinase 1 (NbBAK1), N. benthamiana suppressor of BIR1-1 (NbSOBIR1) and N. benthamiana SGT1 (NbSGT1), but was not suppressed by PiAVR3a or PexRD2. We report the identification of a novel Ascomycete PAMP, RcCDI1, recognized by Solanaceae but not by monocots, which activates cell death through a pathway that is distinct from that triggered by the oomycete PAMP INF1.
D. Parashar, T. Satyanarayana, Front Microbiol 8 493 (2017).
Recombinant chimeric alpha-amylase (Ba-Gt-amy) has been produced extracellularly in Pichia pastoris under AOX promoter. Clones of P. pastoris with multiple gene copies have been generated by multiple transformations and post-transformational vector amplification, which led to 10.7-fold enhancement in alpha-amylase titre as compared to a clone with a copy of the gene. The recombinant P. pastoris integrated eight copies of Ba-Gt-amy in the genome of P. pastoris, as revealed by real time PCR data analysis. Heterologous protein expression as well as mRNA level of Ba-Gt-amy was higher in multi-copy clone than that with single copy. The pure Ba-Gt-amy expressed in P. pastoris is a glycoprotein of 75 kDa, which is optimally active at pH 4.0 and 60 degrees C with T1/2 of 40 min at 70 degrees C. The Kinetic parameters and end product analysis suggested that glycosylation has no effect on catalytic properties of Ba-Gt-amy. The enzyme saccharifies soluble as well as raw starches efficiently and generates maltose and maltooligosaccharides, thus, useful in baking and sugar syrup industries. The strategy for generating multi-copy clones is being reported for the first time, which could be useful in enhancing the production of other recombinant proteins.
A. M. Tran et al., BMC Res Notes 10 148 (2017).
BACKGROUND: Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) is a glycoprotein that has been approved by the FDA for the treatment of neutropenia and leukemia in combination with chemotherapies. Recombinant hGM-CSF is produced industrially using the baker's yeast, Saccharomyces cerevisiae, by large-scale fermentation. The methylotrophic yeast, Pichia pastoris, has emerged as an alternative host cell system due to its shorter and less immunogenic glycosylation pattern together with higher cell density growth and higher secreted protein yield than S. cerevisiae. In this study, we compared the pipeline from gene to recombinant protein in these two yeasts.
RESULTS: Codon optimization in silico for both yeast species showed no difference in frequent codon usage. However, rhGM-CSF expressed from S. cerevisiae BY4742 showed a significant discrepancy in molecular weight from those of P. pastoris X33. Analysis showed purified rhGM-CSF species with molecular weights ranging from 30 to more than 60 kDa. Fed-batch fermentation over 72 h showed that rhGM-CSF was more highly secreted from P. pastoris than S. cerevisiae (285 and 64 mg total secreted protein/L, respectively). Ion exchange chromatography gave higher purity and recovery than hydrophobic interaction chromatography. Purified rhGM-CSF from P. pastoris was 327 times more potent than rhGM-CSF from S. cerevisiae in terms of proliferative stimulating capacity on the hGM-CSF-dependent cell line, TF-1.
CONCLUSION: Our data support a view that the methylotrophic yeast P. pastoris is an effective recombinant host for heterologous rhGM-CSF production.
Q. H. Wang et al., J Asian Nat Prod Res 19 581 (2017).
The scorpion peptide BmK AngM1 was reported to exhibit evident analgesic effect, but its yield by extraction from scorpion venom limits the research and application. The heterologous expression of BmK AngM1 was achieved in Pichia pastoris in our previous study. In order to realize high-level expression of recombinant BmK AngM1 (rBmK AngM1), the gene dosage of BmK AngM1 was optimized in engineered strains. The yield of rBmK AngM1 in the four-copy strain reached up to 100 mg/L, which was further enhanced to 190 mg/L by co-expressing with chaperones of PDI, BiP, and HAC1. Moreover, the yield of rBmK AngM1 was up to 1200 mg/L by high-density fermentation in 10 L fermenter. Finally, 360 mg rBmK AngM1 was purified from 1 L cultures by a two-step purification method. The efficient and convenient techniques presented in this study could facilitate further scale-up for industrial production of rBmK AngM1.
M. Ueda et al., Int J Biol Macromol (2017).
Chitin is the second most abundant biopolymer in nature and is an important resource. In this study, we identified a chitinase gene, named Eisenia fetida-Chitinase (EF-Chi) gene, of 1494 base pairs (bp) that encodes a protein of 498 amino acids as indicated by the corresponding mRNA sequence. The amino acid sequence of EF-Chi was similar to those of chitinases from Eisenia andrei (99%), Branchiostoma floridae (50%) and Oryzias latipes (49%), and a gene encoding mature EF-Chi was expressed in the GS115 strain of Pichia pastoris. The molecular mass of the purified recombinant EF-Chi (rEF-Chi) was estimated to be 60kDa and catalytically important residues of chitinases of the glycoside hydrolase (GH) family 18 were conserved in EF-Chi. The optimal catalytic temperature of rEF-Chi was identified as 60 degrees C, and the hydrolytic product from colloidal chitin was N-acetyl-chitobiose, suggesting that EF-Chi is an exo-type enzyme.
A. Bronikowski, P. L. Hagedoorn, K. Koschorreck, V. B. Urlacher, AMB Express 7 73 (2017).
Laccases have gained significant attention due to their emerging applications including bioremediation, biomass degradation and biofuel cells. One of the prerequisites for the industrial application of laccases is their sufficient availability. However, expression levels of recombinantly expressed laccases are often low. In this study Mrl2, a new laccase from the basidiomycete Moniliophthora roreri, was cloned in Pichia pastoris and produced in an optimized fed-batch process at an exceptionally high yield of 1.05 g l-1. With a redox potential of 0.58 V, Mrl2 belongs to mid-redox potential laccases. However, Mrl2 demonstrated high kcat values of 316, 20, 74, and 36 s-1 towards 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), syringaldazine (SGZ), 2,6-dimethoxyphenol (2,6-DMP) and guaiacol, respectively. Mrl2 remained stable above pH 6 and in the presence of many metal ions, which is important for application in bioremediation. Mrl2 was investigated for the ability to degrade endocrine disrupting chemicals (EDCs) and non-steroidal anti-inflammatory drugs (NSDAIs) at neutral pH value. The enzyme accepted and converted estrone, 17beta-estradiol, estriol, the synthetic contraceptive 17alpha-ethinyl estradiol and bisphenol A at pH 7 faster than high-potential laccases from Trametes versicolor. For example, within 30 min Mrl2 removed more than 90% bisphenol A, 17ss-estradiol, 17alpha-ethinyl estradiol and estriol, respectively. The concentration of the recalcitrant drug diclofenac dropped by 56% after 20 h incubation with Mrl2.
X. D. Wang, T. Jiang, X. W. Yu, Y. Xu, J Ind Microbiol Biotechnol 44 1053 (2017).
Prolyl endopeptidase (PEP) is very useful in various industries, while the high cost of enzyme production remains a major obstacle for its industrial applications. Pichia pastoris has been used for the PEP production; however, the fermentation process has not be investigated and little is known about the impact of excessive PEP production on the host cell physiology. Here, we optimized the nitrogen source to improve the PEP expression level and further evaluated the cellular response including UPR and ERAD. During methanol induction phase the PEP activity (1583 U/L) was increased by 1.48-fold under the optimized nitrogen concentration of NH4+ (300 mmol/L) and casamino acids [1.0% (w/v)] in a 3-L bioreactor. Evaluated by RT-PCR the UPR and ERAD pathways were confirmed to be activated. Furthermore, a strong decrease of ERAD-related gene transcription was observed with the addition of nitrogen source, which contributed to a higher PEP expression level.
G. Oleas, E. Callegari, R. Sepulveda, J. Eyzaguirre, Carbohydr Res 443-444 42 (2017).
The lignocellulolytic fungus, Penicillium purpurogenum, grows on a variety of natural carbon sources, among them sugar beet pulp. Culture supernatants of P. purpurogenum grown on sugar beet pulp were partially purified and the fractions obtained analyzed for esterase activity by zymograms. The bands with activity on methyl umbelliferyl acetate were subjected to mass spectrometry to identify peptides. The peptides obtained were probed against the proteins deduced from the genome sequence of P. purpurogenum. Eight putative esterases thus identified were chosen for future work. Their cDNAs were expressed in Pichia pastoris. The supernatants of the recombinant clones were assayed for esterase activity, and five of the proteins were active against one or more substrates: methyl umbelliferyl acetate, indoxyl acetate, methyl esterified pectin and fluorescein diacetate. Three of those enzymes were purified, further characterized and subjected to a BLAST search. Based on their amino acid sequence and properties, they were identified as follows: RAE1, pectin acetyl esterase (CAZy family CE 12); FAEA, feruloyl esterase (could not be assigned to a CAZy family) and EAN, acetyl esterase (former CAZy family CE 10).
R. Aw, P. F. McKay, R. J. Shattock, K. M. Polizzi, AMB Express 7 70 (2017).
The use of the recombinant expression platform Pichia pastoris to produce pharmaceutically important proteins has been investigated over the past 30 years. Compared to mammalian cultures, expression in P. pastoris is cheaper and faster, potentially leading to decreased costs and process development times. Product yields depend on a number of factors including the secretion signal chosen for expression, which can influence the host cell response to recombinant protein production. VRC01, a broadly neutralising anti-HIV antibody, was expressed in P. pastoris, using the methanol inducible AOX1 promoter for both the heavy and light chains. Titre reached up to 3.05 mug mL-1 in small scale expression. VRC01 was expressed using both the alpha-mating factor signal peptide from Saccharomyces cerevisiae and the murine IgG1 signal peptide. Surprisingly, using the murine IgG1 signal peptide resulted in higher yield of antibody capable of binding gp140 antigen. Furthermore, we evaluated levels of secretory stress compared to the untransformed wild-type strain and show a reduced level of secretory stress in the murine IgG1 signal peptide strains versus those containing the alpha-MF signal peptide. As bottlenecks in the secretory pathway are often the limiting factor in protein secretion, reduced levels of secretory stress and the higher yield of functional antibody suggest the murine IgG1 signal peptide may lead to better protein folding and secretion. This work indicates the possibilities for utilising the murine IgG1 signal peptide for a range of antibodies, resulting in high yields and reduced cellular stress.
M. V. Rocha et al., Vaccine 35 2463 (2017).
Plasmodium vivax is the most widely distributed malaria species and the most prevalent species of malaria in America and Asia. Vaccine development against P. vivax is considered a priority in the global program for the eradication of malaria. Earlier studies have characterized the Apical Membrane Antigen 1 (AMA-1) ectodomain and the C-terminal region (19kDa) of the Merozoite Surface Protein 1 (MSP-1) of P. vivax as immunodominant antigens. Based on this characterization, we designed a chimeric recombinant protein containing both merozoite immunodominant domains (PvAMA166-MSP119). The recombinant PvAMA166-MSP119 was successfully expressed in Pichia pastoris and used to immunize two different mouse strains (BALB/c and C57BL/6) in the presence of the Poly (
I:C) as an adjuvant. Immunization with the chimeric protein induced high antibody titers against both proteins in both strains of mice as detected by ELISA. Antisera also recognized the native proteins expressed on the merozoites of mature P. vivax schizonts. Moreover, this antigen was able to induce IFN-gamma-secreting cells in C57BL/6 mice. These findings indicate that this novel yeast recombinant protein containing PvAMA166 and PvMSP119 is advantageous, because of improved antibody titers and cellular immune response. Therefore, this formulation should be further developed for pre-clinical trials in non-human primates as a potential candidate for a P. vivax vaccine.
M. Mattanovich et al., FEMS Yeast Res 17 (2017).
Mass spectrometry-based metabolomic profiling is a powerful strategy to quantify the concentrations of numerous primary metabolites in parallel. To avoid distortion of metabolite concentrations, quenching is applied to stop the cellular metabolism instantly. For yeasts, cold methanol quenching is accepted to be the most suitable method to stop metabolism, while keeping the cells intact for separation from the supernatant. During this treatment, metabolite loss may occur while the cells are suspended in the quenching solution. An experiment for measuring the time-dependent loss of selected primary metabolites in differently buffered quenching solutions was conducted to study pH and salt concentration-dependent effects. Molecular properties of the observed metabolites were correlated with the kinetics of loss to gain insight into the mechanisms of metabolite leakage. Size and charge-related properties play a major role in controlling metabolite loss. We found evidence that interaction with the cell wall is the main determinant to retain a molecule inside the cell. Besides suggesting an improved quenching protocol to keep loss at a minimum, we could establish a more general understanding of the process of metabolite loss during quenching, which will allow to predict optimal conditions for hitherto not analysed metabolites.
C. Zhan et al., FEMS Yeast Res (2017).
In methylotrophic yeast Pichia pastoris (P. pastoris), the efficient promoter of alcohol oxidase (PAox1) is induced by methanol and repressed by glycerol, but the molecular mechanism is not clear. In this study, the relationship between alcohol oxidase 1 (aox1), methanol expression regulator 1 (mxr1) and glycerol transporter 1 (gt1) was studied. By RT-PCR, it was found that the overexpression of gt1 could increase the glycerol content in cells and repress the expression of mxr1 and aox1, and the deletion of gt1 reduced the glycerol content in cells and promoted the expression of aox1 .The overexpression of mxr1 could repress the expression of gt1, and the deletion of mxr1 could promote the expression of gt1 to some extent. By EMSA, Mxr1 binding sites were found in the promoter of gt1 (PGt1.) (-141 to -138, CCCC), and Mxr1 could regulate the expression of gt1 by binding to PGt1. The relationships among aox1, mxr1 and gt1 revealed here to provide a reference for the understanding of the mechanism of glycerol repression of PAox1.
M. Volpicella et al., Allergy (2017).
Food allergies are recognized as an increasing health concern. Proteins commonly identified as food allergens tend to have one of about 30 different biochemical activities. This leads to the assumption that food allergens must have specific structural features which causes their allergenicity. But these structural features are not completely understood. Uncovering the structural basis of allergenicity would allow improved diagnosis and therapy of allergies and would provide insights for safer food production. The availability of recombinant food allergens can accelerate their structural analysis and benefit specific studies in allergology. Plant chitinases are an example of food allergenic proteins for which structural analysis of allergenicity has only partially been reported. The recombinant maize chitinase, rChiA, was purified from Pichia pastoris extracellular medium by differential precipitation and cation exchange chromatography. Enzyme activity was evaluated by halo-assays and microcalorimetric procedures. rChiA modeling was performed by a two-step procedure, using the Swiss-Model server and Modeller software. Allergenicity of rChiA was verified by immunoblot assays with sera from allergic subjects. rChiA is active in the hydrolysis of glycol chitin and tetra-N-acetylchitotetraose and maintains its activity at high temperatures (70 degrees C) and low pH (pH 3). The molecule is also reactive with IgE from sera of maize-allergic subjects. rChiA is a valuable molecule for further studies on structure-allergenicity relationships and as a tool for diagnosing allergies.
A. Papala et al., Protein Expr Purif 134 25 (2017).
CapG is an actin-binding protein, which is overexpressed in a variety of tumors, i.e. breast, ovarian, pancreatic and lung carcinoma. We successfully expressed human CapG in the wild type strain X-33 of the methylotrophic yeast Pichia pastoris (P. pastoris), which does not express endogenous CapG, in order to characterize this protein in more detail. After mechanical cell lysis, debris was centrifuged and the soluble protein was precipitated with ammonium sulfate. This protein pellet was dialyzed and used for CapG purification. Ca2+-dependent exposure of hydrophobic sites allowed single step and selective elution from a Phenyl Sepharose matrix. 3.5 mg CapG/10 g wet biomass were isolated and showed a Ca2+-sensitive and dose-dependent capping activity of actin in a fluorometric assay. In P. pastoris, CapG is located at actin patches, actin cables and arranges along the budding neck. The proliferation rate and morphology of the yeast cells are not influenced by the interaction of CapG with actin. The modification pattern of human CapG from P. pastoris and human carcinoma cells is highly similar. We validated most of the known post-translational modifications and found three new phosphorylation and nine new acetylation sites by mass spectrometry. The N-terminus is acetylated or truncated. Truncated CapG is not phosphorylated at the residues S10, T212 and S337. First mutagenesis experiments indicate an N-terminal acetylation dependent C-terminal phosphorylation.
X. Zhao, H. Hong, Z. Wu, Protein Expr Purif 133 132 (2017).
In order to achieve efficient extracellular expression of Sortase A (SrtA), various strategies in Pichia pastoris system were applied in this study. Among different constructed recombinant strains, the SMD1168 strain integrated 5.7 copies of srtA gene under control of AOX1 promoter was proved to be the best strain for the extracellular SrtA expression. After the optimization of fermentation conditions (induction 72 h at 28 degrees C, initial pH 6.0, supplemented with 1.5% methanol), the highest yield and activity of extracellular SrtA reached 97.8 mg/L and 131.9 U/mL at the shake-flask level, respectively. This is the first report on the efficient secretory expression of SrtA in P. pastoris and the yield of SrtA is the maximum compared with previous reports. In addition, the transpeptidation activity of extracellular SrtA was confirmed by the successful immobilization of enhanced green fluorescent protein (EGFP) onto Gly3-polystyrene beads.
Q. Zheng et al., Mol Oncol 11 584 (2017).
CD19 is expressed on normal and neoplastic B cells and is a promising target for immunotherapy. However, there is still an unmet need to further develop novel therapeutic drugs for the treatment of the refractory/relapsing human CD19+ tumors. We have developed a diphtheria toxin-based anti-human CD19 immunotoxin for targeting human CD19+ tumors. We have constructed three isoforms of the CD19 immunotoxin: monovalent, bivalent, and foldback diabody. In vitro binding affinity and efficacy analysis demonstrated that the bivalent isoform had the highest binding affinity and in vitro efficacy. The in vivo efficacy of the CD19 immunotoxins was assessed using human CD19+ JeKo-1 tumor-bearing NOD/SCID IL-2 receptor gamma-/- (NSG) mouse model. In these animals, CD19 immunotoxins significantly prolonged the median survival from 31 days in controls to 34, 36, and 40 days in animals receiving the monovalent isoform, foldback diabody isoform, and bivalent isoform, respectively. The bivalent CD19 immunotoxin is a promising therapeutic drug candidate for targeting relapsing/refractory human CD19+ tumors.
J. W. Moser et al., Microb Cell Fact 16 49 (2017).
BACKGROUND: Pichia pastoris is a widely used eukaryotic expression host for recombinant protein production. Adaptive laboratory evolution (ALE) has been applied in a wide range of studies in order to improve strains for biotechnological purposes. In this context, the impact of long-term carbon source adaptation in P. pastoris has not been addressed so far. Thus, we performed a pilot experiment in order to analyze the applicability and potential benefits of ALE towards improved growth and recombinant protein production in P. pastoris.
RESULTS: Adaptation towards growth on methanol was performed in replicate cultures in rich and minimal growth medium for 250 generations. Increased growth rates on these growth media were observed at the population and single clone level. Evolved populations showed various degrees of growth advantages and trade-offs in non-evolutionary growth conditions. Genome resequencing revealed a wide variety of potential genetic targets associated with improved growth performance on methanol-based growth media. Alcohol oxidase represented a mutational hotspot since four out of seven evolved P. pastoris clones harbored mutations in this gene, resulting in decreased Aox activity, despite increased growth rates. Selected clones displayed strain-dependent variations for AOX-promoter based recombinant protein expression yield. One particularly interesting clone showed increased product titers ranging from a 2.5-fold increase in shake flask batch culture to a 1.8-fold increase during fed batch cultivation.
CONCLUSIONS: Our data indicate a complex correlation of carbon source, growth context and recombinant protein production. While similar experiments have already shown their potential in other biotechnological areas where microbes were evolutionary engineered for improved stress resistance and growth, the current dataset encourages the analysis of the potential of ALE for improved protein production in P. pastoris on a broader scale.
Y. Lu et al., Sci Rep 7 44719 (2017).
X. Liu et al., J Microbiol Biotechnol (2017).
Vesicular stomatitis virus G glycoprotein (VSV-G) has been widely used for pseudotyping retroviral, lentiviral and artificial viral vectors. The objective of this study was to establish a potential approach for large scale production of VSV-G. To this end, VSV-G was cloned with a N-terminal His-tag into Pichia pastoris expression vector pPIC3.5K. Three clones (Muts) containing the VSV-G expression cassette were identified by PCR. All clones proliferated normally in expansion medium, while the proliferation was reduced significantly under induction conditions. VSV-G protein was detected in cell lysates by Western Blot, and the highest expression level was observed at 96 h post induction. VSV-G could also be obtained from the condition medium of yeast protoplasts. Further, VSV-G could be incorporated into Ad293 cells and was able to induce cell fusion, leading to the transfer of cytoplasmic protein. Finally, VSV-G mediated DNA transfection was assayed by flow cytometry and luciferase measurement. Incubation of VSV-G lysate with pGL3-control DNA complex increased luciferase activity in Ad293 and HeLa cells by about 3-fold. Likewise, incubation of VSV-G lysate with pCMV-DsRed DNA complex improved transfection efficiency into Ad293 by 10% and HeLa cells by about 1-fold. In conclusions, these results demonstrated that VSV-G could be produced from Pichia pastoris with bio-functionalities, demonstrating large scale production of the viral glycoprotein is feasible.
E. Camara et al., Sci Rep 7 44302 (2017).
The methanol-regulated alcohol oxidase promoter (PAOX1) of Pichia pastoris is one of the strongest promoters for heterologous gene expression in this methylotrophic yeast. Although increasing gene dosage is one of the most common strategies to increase recombinant protein productivities, the increase of gene dosage of Rhizopus oryzae lipase (ROL) in P. pastoris has been previously shown to reduce cell growth, lipase production and substrate consumption in high-copy strains. To better assess that physiological response, transcriptomics analysis was performed of a subset of strains with 1 to 15 ROL copies. The macroscopic physiological parameters confirm that growth yield and carbon uptake rate are gene dosage dependent, and were supported by the transcriptomic data, showing the impact of increased dosage of AOX1 promoter-regulated expression cassettes on P. pastoris physiology under steady methanolic growth conditions. Remarkably, increased number of cassettes led to transcription attenuation of the methanol metabolism and peroxisome biogenesis in P. pastoris, concomitant with reduced secretion levels of the heterologous product. Moreover, our data also point to a block in ROL mRNA translation in the higher ROL-copies constructs, while the low productivities of multi-copy strains under steady growth conditions do not appear to be directly related to UPR and ERAD induction.
A. Dilokpimol et al., N Biotechnol 37 200 (2017).
A feruloyl esterase (FAE) from Aspergillus niger N402, FaeC was heterologously produced in Pichia pastoris X-33 in a yield of 10mg/L. FaeC was most active at pH 7.0 and 50 degrees C, and showed broad substrate specificity and catalyzed the hydrolysis of methyl 3,4-dimethoxycinnamate, ethyl ferulate, methyl ferulate, methyl p-coumarate, ethyl coumarate, methyl sinapate, and methyl caffeate. The enzyme released both ferulic acid and p-coumaric acid from wheat arabinoxylan and sugar beet pectin (up to 3mg/g polysaccharide), and acted synergistically with a commercial xylanase increasing the release of ferulic acid up to six-fold. The expression of faeC increased over time in the presence of feruloylated polysaccharides. Cinnamic, syringic, caffeic, vanillic and ferulic acid induced the expression of faeC. Overall expression of faeC was very low in all tested conditions, compared to two other A. niger FAE encoding genes, faeA and faeB. Our data showed that the fae genes responded differently towards the feruloylated polysaccharides and tested monomeric phenolic compounds suggesting that the corresponding FAE isoenzymes may target different substrates in a complementary manner. This may increase the efficiency of the degradation of diverse plant biomass.
G. Yang et al., J Mol Endocrinol 58 179 (2017).
CTRP9 is a member of the C1q/TNF-related protein (CTRP) superfamily and has been studied in mammals, whereas the comparative studies of CTRP9 in non-mammalian species are still absent. In this study, ctrp9 was isolated and characterized from the orange-spotted grouper (Epinephelus coioides). The full-length cDNA of ctrp9 was 1378 bp in size with an ORF (open reading frame) of 1020 bp that encodes a 339 amino acid pre-pro hormone. The mRNA expression of ctrp9 showed a rather high level in the kidney and brain, but a low level in other tissues. Furthermore, the mRNA expression of ctrp9 decreased significantly in the liver after fasting for 7 days and restored to the normal levels after refeeding. In contrast, the ctrp9 mRNA level increased in the hypothalamus after fasting. The recombinant gCtrp9 (globular Ctrp9) was prepared using the Pichia pastoris expression system and was verified by Western blot as well as mass spectrometry assays. In the primary hepatocytes culture, the recombinant gCtrp9 could inhibit the glucose production after 12-h treatment. After i.p. (intraperitoneal) injection with recombinant gCtrp9, in hypothalamus, mRNA expression levels of npy and orexin (orexigenic factors) decreased, whereas the expression levels of crh and pomc (anorexigenic factors) increased. Moreover, i.p. injection with the recombinant gCtrp9 could reduce the serum concentrations of glucose, TG and low-density lipoprotein cholesterol but increase the content of high-density lipoprotein cholesterol. Our studies for the first time unveil the structure of Ctrp9 and its potential role as a regulatory factor of metabolism and food intake in teleost.
H. Maresova et al., Folia Microbiol (Praha) (2017).
This study deals with the potential of Pichia pastoris X-33 for the production of penicillin G acylase (PGAA) from Achromobacter sp. CCM 4824. Synthetic gene matching the codon usage of P. pastoris was designed for intracellular and secretion-based production strategies and cloned into vectors pPICZ and pPICZalpha under the control of AOX1 promoter. The simple method was developed to screen Pichia transformants with the intracellularly produced enzyme. The positive correlation between acylase production and pga gene dosage for both expression systems was demonstrated in small scale experiments. In fed-batch bioreactor cultures of X-33/PENS2, an extracellular expression system, total PGAA expressed from five copies reached 14,880 U/L of an active enzyme after 142 h; however, 60% of this amount retained in the cytosol. The maximum PGAA production of 31,000 U/L was achieved intracellularly from nine integrated gene copies of X-33/PINS2 after 90 h under methanol induction. The results indicate that in both expression systems the production level of PGAA is similar but there is a limitation in secretion efficiency.
H. Ampah-Korsah et al., BMC Plant Biol 17 61 (2017).
BACKGROUND: Aquaporins (AQPs) are integral membrane proteins that facilitate transport of water and/or other small neutral solutes across membranes in all forms of life. The X Intrinsic Proteins (XIPs) are the most recently recognized and the least characterized aquaporin subfamily in higher plants. XIP1s have been shown to be impermeable to water but permeable to boric acid, glycerol, hydrogen peroxide and urea. However, uncertainty regarding the determinants for selectivity and lack of an activity that is easy to quantify have hindered functional investigations. In an effort to resolve these issues, we set out to introduce water permeability in Nicotiana benthamiana XIP1;1alpha (NbXIP1;1alpha), by exchanging amino acid residues of predicted alternative aromatic/arginine (ar/R) selectivity filters of NbXIP1;1alpha for residues constituting the water permeable ar/R selectivity filter of AtTIP2;1.
RESULTS: Here, we present functional results regarding the amino acid substitutions in the putative filters as well as deletions in loops C and D of NbXIP1;1alpha. In addition, homology models were created based on the high resolution X-ray structure of AtTIP2;1 to rationalize the functional properties of wild-type and mutant NbXIP1;1alpha. Our results favour Thr 246 rather than Val 242 as the residue at the helix 5 position in the ar/R filter of NbXIP1;1alpha and indicate that the pore is not occluded by the loops when heterologously expressed in Pichia pastoris. Moreover, our results show that a single amino acid substitution in helix 1 (L79G) or in helix 2 (I102H) is sufficient to render NbXIP1;1alpha water permeable. Most of the functional results can be rationalized from the models based on a combination of aperture and hydrophobicity of the ar/R filter.
CONCLUSION: The water permeable NbXIP1;1alpha mutants imply that the heterologously expressed proteins are correctly folded and offer means to explore the structural and functional properties of NbXIP1;1alpha. Our results support that Thr 246 is part of the ar/R filter. Furthermore, we suggest that a salt bridge to an acidic residue in helix 1, conserved among the XIPs in clade B, directs the orientation of the arginine in the ar/R selectivity filter and provides a novel approach to tune the selectivity of AQPs.
P. M. Swe et al., PLoS Negl Trop Dis 11 e0005437 (2017).
BACKGROUND: On a global scale scabies is one of the most common dermatological conditions, imposing a considerable economic burden on individuals, communities and health systems. There is substantial epidemiological evidence that in tropical regions scabies is often causing pyoderma and subsequently serious illness due to invasion by opportunistic bacteria. The health burden due to complicated scabies causing cellulitis, bacteraemia and sepsis, heart and kidney diseases in resource-poor communities is extreme. Co-infections of group A streptococcus (GAS) and scabies mites is a common phenomenon in the tropics. Both pathogens produce multiple complement inhibitors to overcome the host innate defence. We investigated the relative role of classical (CP), lectin (LP) and alternative pathways (AP) towards a pyodermic GAS isolate 88/30 in the presence of a scabies mite complement inhibitor, SMSB4. METHODOLOGY/
PRINCIPAL FINDINGS: Opsonophagocytosis assays in fresh blood showed baseline immunity towards GAS. The role of innate immunity was investigated by deposition of the first complement components of each pathway, specifically C1q, FB and MBL from normal human serum on GAS. C1q deposition was the highest followed by FB deposition while MBL deposition was undetectable, suggesting that CP and AP may be mainly activated by GAS. We confirmed this result using sera depleted of either C1q or FB, and serum deficient in MBL. Recombinant SMSB4 was produced and purified from Pichia pastoris. SMSB4 reduced the baseline immunity against GAS by decreasing the formation of CP- and AP-C3 convertases, subsequently affecting opsonisation and the release of anaphylatoxin. CONCLUSIONS/
SIGNIFICANCE: Our results indicate that the complement-inhibitory function of SMSB4 promotes the survival of GAS in vitro and inferably in the microenvironment of the mite-infested skin. Understanding the tripartite interactions between host, parasite and microbial pathogens at a molecular level may serve as a basis to develop improved intervention strategies targeting scabies and associated bacterial infections.
O. A. Asojo et al., PLoS Negl Trop Dis 11 e0005374 (2017).
BACKGROUND: Immunity to the sand fly salivary protein SALO (Salivary Anticomplement of Lutzomyia longipalpis) protected hamsters against Leishmania infantum and L. braziliensis infection and, more recently, a vaccine combination of a genetically modified Leishmania with SALO conferred strong protection against L. donovani infection. Because of the importance of SALO as a potential component of a leishmaniasis vaccine, a plan to produce this recombinant protein for future scale manufacturing as well as knowledge of its structural characteristics are needed to move SALO forward for the clinical path. METHODOLOGY/
PRINCIPAL FINDINGS: Recombinant SALO was expressed as a soluble secreted protein using Pichia pastoris, rSALO(P), with yields of 1g/L and >99% purity as assessed by SEC-MALS and SDS-PAGE. Unlike its native counterpart, rSALO(P) does not inhibit the classical pathway of complement; however, antibodies to rSALO(P) inhibit the anti-complement activity of sand fly salivary gland homogenate. Immunization with rSALO(P) produces a delayed type hypersensitivity response in C57BL/6 mice, suggesting rSALO(P) lacked anti-complement activity but retained its immunogenicity. The structure of rSALO(P) was solved by S-SAD at Cu-Kalpha to 1.94 A and refined to Rfactor 17%. SALO is ~80% helical, has no appreciable structural similarities to any human protein, and has limited structural similarity in the C-terminus to members of insect odorant binding proteins. SALO has three predicted human CD4+ T cell epitopes on surface exposed helices. CONCLUSIONS/
SIGNIFICANCE: The results indicate that SALO as expressed and purified from P. pastoris is suitable for further scale-up, manufacturing, and testing. SALO has a novel structure, is not similar to any human proteins, is immunogenic in rodents, and does not have the anti-complement activity observed in the native salivary protein which are all important attributes to move this vaccine candidate forward to the clinical path.
M. A. Khan et al., Biosci Biotechnol Biochem 81 1114 (2017).
Previously, we have reported cloning of human epidermal growth factor gene from Huh-7 cells and its extracellular expression in Pichia pastoris. The presented work is a detailed report regarding molecular characterization of Huh-7 cells-derived hEGF expressed in Pichia pastoris with special reference to its glycosylation profiling and bioactivity studies. Densitometric scanning of SDS-PAGE separated extracellular proteins from hEGF recombinant Pichia pastoris strain indicated that about 84% of the extracellular proteins were glycosylated. Size exclusion chromatography using Superdex 75 prep grade column was successfully utilized to separate fractions containing glycosylated and non-glycosylated extracellular proteins. In dot blot assay, hEGF was detected in both glycosylated and non-glycosylated fractions. Bioactivity assays revealed that both glycosylated and non-glycosylated fractions were bioactive as determined by cell viability assay. It was also observed that hEGF present in non-glycosylated fraction was relatively more bioactive than hEGF present in glycosylated fraction.
X. Q. Yang et al., J Agric Food Chem 65 2337 (2017).
On the basis of prior work, cytochrome P450 CYP9A61 was found to be enriched in fat bodies and during feeding stages, and transcription was induced by lambda-cyhalothrin in Cydia pomonella. In this study, recombinant CYP9A61 was expressed in Escherichia coli and Pichia pastoris, and its biochemical properties were investigated. Substrate saturation curves and biochemical properties revealed that, in the presence of glycosylation, the yeast-secreted CYP9A61 exhibited a higher affinity for the substrate p-nitroanisole and was found to be more stable at certain pHs and temperatures than bacterially produced CYP9A61. Half-inhibitory concentrations (IC50) of three synthetic pyrethroids on both the bacterium- and yeast-expressed CYP9A61 suggested that recombinant CYP9A61 expressed in different hosts exhibits different inhibition properties. Taken together, our findings show that yeast-expressed CYP9A61 exhibits enzyme activity that is better than that expressed in bacteria and might be used for further metabolism assays to reveal the insecticide-detoxifying role of CYP9A61 in C. pomonella.
Z. Li et al., J Ind Microbiol Biotechnol 44 973 (2017).
Prenylated quinones, especially menaquinones, have significant physiological activities, but are arduous to synthesize efficiently. Due to the relaxed aromatic substrate specificity and prenylation regiospecificity at the ortho- site of the phenolic hydroxyl group, the aromatic prenyltransferase NovQ from Streptomyces may be useful in menaquinone synthesis from menadione. In this study, NovQ was overexpressed in Pichia pastoris. After fermentation optimization, NovQ production increased by 1617%. Then the different effects of metal ions, detergents and pH on the activity of purified NovQ were investigated to optimize the prenylation reaction. Finally, purified NovQ and cells containing NovQ were used for menadione prenylation in vitro and in vivo, respectively. Menaquinone-1 (MK-1) was detected as the only product in vitro with gamma,gamma-dimethylallyl pyrophosphate and menadione hydroquinol substrates. MK-3 at a concentration of 90.53 mg/L was detected as the major product of whole cell catalysis with 3-methyl-2-buten-1-ol and menadione hydroquinol substrates. This study realized whole cell catalysis converting menadione to menaquinones.
Z. Cheng et al., J Biosci Bioeng 123 665 (2017).
Endo-polygalacturonases play an important role on depectinization in fruit juices industry. A putative endo-polygalacturonase gene PoxaEnPG28A was cloned from Penicillium oxalicum CZ1028. PoxaEnPG28A consisted of a putative signal peptide and a catalytic domain belonging to glycoside hydrolase family 28, and it shared 72% identity with that of a functionally characterized endo-polygalacturonase from Trichoderma harzianum. Gene PoxaEnPG28A was successfully expressed in Pichia pastoris with a high yield of 1828.7 U/mL. The purified recombinant enzyme PoxaEnPG28A hydrolyzed polygalacturonic acid in endo-manner releasing oligo-galacturonates. PoxaEnPG28A showed maximal activity at pH 5.5 and 55 degrees C, and was stable between pH 3.0 to 10.0 and below 45 degrees C. The kinetic constants Km and Vmax of PoxaEnPG28A were calculated as 1.57 g/L and 14,641.29 U/mg, respectively. PoxaEnPG28A significantly improved the yields of fruit juices from banana, plantain, papaya, pitaya and mango. The high production level of the recombinant enzyme PoxaEnPG28A by P. pastoris and remarkable catalytic activity of PoxaEnPG28A toward five kinds of fruit juices made the enzyme a potential application in agriculture and food industries.
U. Obst, T. K. Lu, V. Sieber, ACS Synth Biol 6 1016 (2017).
Yeasts are powerful eukaryotic hosts for the production of recombinant proteins due to their rapid growth to high cell densities and ease of genetic modification. For large-scale industrial production, secretion of a protein offers the advantage of simple and efficient downstream purification that avoids costly cell rupture, denaturation and refolding. The methylotrophic yeast Pichia pastoris (Komagataella phaffi) is a well-established expression host that has the ability to perform post-translational modifications and is generally regarded as safe (GRAS). Nevertheless, optimization of protein secretion in this host remains a challenge due to the multiple steps involved during secretion and a lack of genetic tools to tune this process. Here, we developed a toolkit of standardized regulatory elements specific for Pichia pastoris allowing the tuning of gene expression and choice of protein secretion tag. As protein secretion is a complex process, these parts are compatible with a hierarchical assembly method to enable the generation of large and diverse secretion libraries in order to explore a wide range of secretion constructs, achieve successful secretion, and better understand the regulatory factors of importance to specific proteins of interest. To assess the performance of these parts, we built and characterized the expression and secretion efficiency of 124 constructs that combined different regulatory elements with two fluorescent reporter proteins (RFP, yEGFP). Intracellular expression from our promoters was comparatively independent of whether RFP or yEGFP, and whether plasmid-based expression or genomically integrated expression, was used. In contrast, secretion efficiency significantly varied for different genes expressed using identical regulatory elements, with differences in secretion efficiency of >10-fold observed. These results highlight the importance of generating diverse secretion libraries when searching for optimal expression conditions, and demonstrate that our toolkit is a valuable asset for the creation of efficient microbial cell factories.
J. Wang et al., J Agric Food Chem 65 2730 (2017).
An endo-polygalacturonase gene (pga-zj5a) was cloned by reverse transcription from cDNAs synthesized from Aspergillus niger ZJ5 total RNA. The open reading frame of pga-zj5a was 1089 base pairs encoding 362 amino acids. Pga-zj5a lacking a signal peptide sequence was successfully amplified using A. niger ZJ5 cDNA as the template and was ligated into the pPIC9 vector. The resulting plasmid was transformed into competent cells of Pichia pastoris GS115 for heterologous expression. The polygalacturonase showed a maximum activity level of 10436 U/mL in the culture supernatant from a 3 L fermenter. Assays of enzymatic properties showed that the optimal pH and temperature of the recombinant PGA-ZJ5A were 4.5 and 40 degrees C, respectively. PGA-ZJ5A was effective in pear juice clarification, increased the volume of pear juice by 41.8%, and improved its light transmittance 3-fold.
S. Ma et al., Microb Cell Fact 16 37 (2017).
BACKGROUND: Cellobiose dehydrogenase (CDH) is an extracellular enzyme produced by lignocellulolytic fungi. cdh gene expression is high in cellulose containing media, but relatively low CDH concentrations are found in the supernatant of fungal cultures due to strong binding to cellulose. Therefore, heterologous expression of CDH in Pichia pastoris was employed in the last 15 years, but the obtained enzymes were over glycosylated and had a reduced specific activity.
RESULTS: We compare the well-established CDH expression host P. pastoris with the less frequently used hosts Escherichia coli, Aspergillus niger, and Trichoderma reesei. The study evaluates the produced quantity and protein homogeneity of Corynascus thermophilus CDH in the culture supernatants, the purification, and finally compares the enzymes in regard to cofactor loading, glycosylation, catalytic constants and thermostability.
CONCLUSIONS: Whereas E. coli could only express the catalytic dehydrogenase domain of CDH, all eukaryotic hosts could express full length CDH including the cytochrome domain. The CDH produced by T. reesei was most similar to the CDH originally isolated from the fungus C. thermophilus in regard to glycosylation, cofactor loading and catalytic constants. Under the tested experimental conditions the fungal expression hosts produce CDH of superior quality and uniformity compared to P. pastoris.
G. R. Rudramurthy, P. P. Sengupta, M. Ligi, H. Rahman, Biologicals 46 148 (2017).
The present study is aimed at the development of inhibition ELISA (I-ELISA) exploring monoclonal antibodies (MAbs) and recombinant invariant surface glycoprotein. The extracellular domain (ED) of invariant surface glycoprotein (ISG-75) from Trypanosoma evasni has been heterologously expressed in Pichia pastoris (X-33). The recombinant ISG-75 (rISG-75ED) was characterized by immunoblot and ELISA, followed by the production of MAbs against rISG-75ED. The MAbs were characterized by immunoblot and then explored in the development of I-ELISA for the detection of surra. The diagnostic potential of the developed test has been evaluated using 1192 field sera sample including cattle, buffalo, donkey, horse and camel. The statistical analysis of the data showed optimum combination of diagnostic sensitivity and specificity at 98.8% and 99.2% respectively, with cut-off percentage inhibition (PI) value of >45. The Cohen's kappa coefficient of agreement was found to be 0.98. Hence, the diagnostic test developed in the present study can be exploited as a potential and reliable tool in the serodiagnosis and surveillance of surra in animals.
L. Marsalek et al., Biotechnol J 12 (2017).
The methylotrophic yeast Pichia pastoris (Komagataella spp.) is a popular microbial host for the production of recombinant proteins. Previous studies have shown that mis-sorting to the vacuole can be a bottleneck during production of recombinant secretory proteins in yeast, however, no information was available for P. pastoris. In this work the authors have therefore generated vps (vacuolar protein sorting) mutant strains disrupted in genes involved in the CORVET (class C core vacuole/endosome tethering) complex at the early stages of endosomal sorting. Both Deltavps8 and Deltavps21 strains contained lower extracellular amounts of heterologous carboxylesterase (CES) compared to the control strain, which could be attributed to a high proteolytic activity present in the supernatants of CORVET engineered strains due to rerouting of vacuolar proteases. Serine proteases were identified to be responsible for this proteolytic degradation by liquid chromatography-mass spectrometry and protease inhibitor assays. Deletion of the major cellular serine protease Prb1 in Deltavps8 and Deltavps21 strains did not only rescue the extracellular CES levels, but even outperformed the parental CES strain (56 and 80% higher yields, respectively). Further deletion of Ybr139W, another serine protease, did not show a further increase in secretion levels. Higher extracellular CES activity and low proteolytic activity were detected also in fed batch cultivation of Deltavps21Deltaprb1 strains, thus confirming that modifying early steps in the vacuolar pathway has a positive impact on heterologous protein secretion.
K. Salamin et al., Appl Microbiol Biotechnol 101 4129 (2017).
Prolyl peptidases of the MEROPS S28 family are of particular interest because they are key enzymes in the digestion of proline-rich peptides. A BLAST analysis of the Aspergillus oryzae genome revealed sequences coding for four proteases of the S28 family. Three of these proteases, AoS28A, AoS28B, and AoS28C, were previously characterized as acidic prolyl endopeptidases. The fourth protease, AoS28D, showed high sequence divergence with other S28 proteases and belongs to a phylogenetically distinct cluster together with orthologous proteases from other Aspergillus species. The objective of the present paper was to characterize AoS28D protease in terms of substrate specificity and activity. AoS28D produced by gene overexpression in A. oryzae and in Pichia pastoris was a 70-kDa glycoprotein with a 10-kDa sugar moiety. In contrast with other S28 proteases, AoS28D did not hydrolyze internal Pro-Xaa bonds of several tested peptides. Similarly, to human lysosomal Pro-Xaa carboxypeptidase, AoS28D demonstrated selectivity for cleaving C-terminal Pro-Xaa bonds which are resistant to carboxypeptidases of the S10 family concomitantly secreted by A. oryzae. Therefore, AoS28D could act in synergy with these enzymes during sequential degradation of a peptide from its C-terminus.
M. Takacs, O. V. Makhlynets, P. L. Tolbert, I. V. Korendovych, Protein Eng Des Sel 30 381 (2017).
Biofuels are an important tool for the reduction of carbon dioxide and other greenhouse emissions. NAD+-dependent formate dehydrogenase has been previously shown to be capable of the electrochemical reduction of carbon dioxide into formate, which can be ultimately converted to methanol. We established that a functional enzyme, tagged for immobilization, could be continuously secreted by Pichia pastoris. The protein can be easily separated from the growth media and its activity remains constant over an extended period of time. This is an important first step in creating a self-sustaining system capable of producing biofuels with minimal resources and space required.
J. Gao et al., Enzyme Microb Technol 99 32 (2017).
1,4-beta-Endoglucanase is one of the most important biocatalysts in modern industries. Here, a glycoside hydrolase (GH) family 45 endoglucanase from thermophilic fungus Theilavia terrestris (TtCel45A) was expressed in Pichia pastoris. The recombinant protein shows optimal activity at 60 degrees C, pH 4-5. The enzyme exhibits extraordinary thermostability that more than 80% activity was detected after heating at 80 degrees C for 2.5h. The high resolution crystal structures of apo-form enzyme and that in complex with cellobiose and cellotetraose were solved to 1.36-1.58A. The protein folds into two overall regions: one is a six-stranded beta-barrel, and the other one consists of several extended loops. Between the two regions lies the substrate-binding channel, which is an open cleft spanning across the protein surface. A continuous substrate-binding cleft from subsite -4 to +3 were clearly identified in the complex structures. Notably, the flexible V-VI loop (113Gly-114Gly-115Asp-116Leu-117Gly-118Ser) is found to open in the presence of -1 sugar, with D115 and L116 swung away to yield a space to accommodate the catalytic acid D122 and the 2,5B boat conformation of -1 sugar during transition state. Collectively, we characterized the enzyme properties of P. pastoris-expressed TtCel45A and solved high-resolution crystal structures of the enzyme. These results are of great interests in industrial applications and provide new insights into the fundamental understanding of enzyme catalytic mechanism of GH45 endoglucanases.
L. D. Picotto et al., Protein Expr Purif 132 124 (2017).
The glycoprotein (G-protein) of rabies virus is responsible for viral attachment to the host cell surface and induces virus neutralization antibodies. In the present study, the G-protein gene of rabies virus CVS strain was cloned, sequenced and expressed in the yeast, Pichia pastoris, as a secreted protein, using a simplified DO-stat control feeding strategy. This strategy involves the addition of methanol when the dissolved oxygen (DO) level rises above the setpoint avoiding methanol accumulation and oxygen limitation. The G-protein expression was evaluated by SDS-PAGE, ELISA, and western blot assays. Like native G-protein, the recombinant G-protein was found reactive when it was challenged against specific antibodies. The data indicate that the recombinant G-protein can be easily expressed and isolated, and may be useful as a safe source in the production of diagnostic kits and subunit vaccines to prevent rabies.
H. Hu et al., J Microbiol Biotechnol 27 775 (2017).
A neutral xylanase (CcXyn) was identified from Coprinus cinereus. It has a single GH10 catalytic domain with a basic amino acid-rich extension (PVRRK) at the C-terminus. In this study, the wild-type (CcXyn) and C-terminus-truncated xylanase (CcXyn-Delta5C) were heterologously expressed in Pichia pastoris and their characteristics were comparatively analyzed with aims to examine the effect of this extension on the enzyme function. The circular dichorism analysis indicated that both enzymes in general had a similar structure, but CcXyn-Delta5C contained less alpha-helices (42.9%) and more random coil contents (35.5%) than CcXyn (47.0% and 32.8%, respectively). Both enzymes had the same pH (7.0) and temperature (45 degrees C) optima, and similar substrate specificity on different xylans. They all hydrolyzed beechwood xylan primarily to xylobiose and xylotriose. The amounts of xylobiose and xylotriose accounted for 91.5% and 92.2% (w/w) of total xylooligosaccharides (XOS) generated from beechwood by CcXyn and CcXyn-Delta5C, respectively. However, truncation of the C-terminal 5-amino-acids extension significantly improved the thermostability, SDS resistance, and pH stability at pH 6.0-9.0. Furthermore, CcXyn-Delta5C exhibited a much lower Km value than CcXyn (0.27 mg/ml vs 0.83 mg/ml), and therefore, the catalytic efficiency of CcXyn-Delta5C was 2.4-times higher than that of CcXyn. These properties make CcXyn-Delta5C a good model for the structure-function study of (alpha/beta)8-barrel-folded enzymes and a promising candidate for various applications, especially in the detergent industry and XOS production.
O. H. Kim et al., Toxicon 129 153 (2017).
Recombinant batroxobin is a thrombin-like enzyme of Bothrops atrox moojeni venom. To evaluate its toxicological effect, it was highly expressed in Pichia pastorisand successfully purified to homogeneity from culture broth supernatant following Good Manufacturing Practice (GMP). The maximum tolerated dose of the recombinant batroxobin was examined in Sprague-Dawley (SD) rat and Beagle dogs following Good Laboratory Practice (GLP) regulations. The approximate lethal dose of recombinant batroxobin was 10 National Institute of Health (NIH) u/kg in male and female rats. Slight test substance-related effects were clearly in male and female dogs at more than 10 NIH u/kg. The maximum tolerated dose (MTD) was considered to be greater than 30 NIH u/kg in dogs. To investigate the repeated dose toxicity of batroxobin, the test item was intravenously administered to groups of SD rat and Beagle dog every day for 4 weeks. We observed that all animals survived the duration of the study without any effects on their mortality. There were no effects in both rats and dogs regarding their clinical signs, body weight, food consumption, ophthalmological examination, urinalysis, hematology, clinical chemistry, organ weightand gross post mortem examinations. The no adverse effect level (NOAEL) of recombinant batroxobin for both males and females is considered to be greater than 2.5 NIH u/kgin rats and 1 NIH u/kg in dogs, respectively. No toxic effects were noted in target organs. In conclusion, these results show a favorable preclinical profile and may contribute clinical development of recombinant batroxobin.
G. D. Tredwell et al., J Ind Microbiol Biotechnol 44 413 (2017).
Heterologous protein production in the yeast Pichia pastoris can be limited by biological responses to high expression levels; the unfolded protein response (UPR) is a key determinant of the success of protein production in this organism. Here, we used untargeted NMR metabolic profiling (metabolomics) of a number of different recombinant strains, carried out in a miniaturized format suitable for screening-level experiments. We identified a number of metabolites (from both cell extracts and supernatants) which correlated well with UPR-relevant gene transcripts, and so could be potential biomarkers for future high-throughput screening of large numbers of P. pastoris clones.
A. Stachyra et al., Virus Res 232 41 (2017).
Highly pathogenic avian influenza viruses cause severe disease and huge economic losses in domestic poultry and might pose a serious threat to people because of the high mortality rates in case of an accidental transmission to humans. The main goal of this work was to evaluate the immune responses and hemagglutination inhibition potential elicited by a combined DNA/recombinant protein prime/boost vaccination compared to DNA/DNA and protein/protein regimens in chickens. A plasmid encoding hemagglutinin (HA) from the A/swan/Poland/305-135V08/2006 (H5N1) virus, or the recombinant HA protein produced in Pichia pastoris system, both induced H5 HA-specific humoral immune responses in chickens. In two independent experiments, anti-HA antibodies were detected in sera collected two weeks after the first dose and the response was enhanced by the second dose of a vaccine, regardless of the type of subunit vaccine (DNA or recombinant protein) administered. The serum collected from chickens two weeks after the second dose was characterized by three types of assays: indirect ELISA, hemagglutination inhibition (HI) and a diagnostic test based on H5 antibody competition. Although the indirect ELISA failed to detect superiority of any of the three vaccine regimens, the other two tests clearly indicated that priming of chickens with the DNA vaccine significantly enhanced the protective potential of the recombinant protein vaccine produced in P. pastoris.
P. G. Landim et al., Biochimie 135 89 (2017).
A cowpea class I chitinase (VuChiI) was expressed in the methylotrophic yeast P. pastoris. The recombinant protein was secreted into the culture medium and purified by affinity chromatography on a chitin matrix. The purified chitinase migrated on SDS-polyacrylamide gel electrophoresis as two closely-related bands with apparent molecular masses of 34 and 37 kDa. The identity of these bands as VuChiI was demonstrated by mass spectrometry analysis of tryptic peptides and N-terminal amino acid sequencing. The recombinant chitinase was able to hydrolyze colloidal chitin but did not exhibit enzymatic activity toward synthetic substrates. The highest hydrolytic activity of the cowpea chitinase toward colloidal chitin was observed at pH 5.0. Furthermore, most VuChiI activity (approximately 92%) was retained after heating to 50 degrees C for 30 min, whereas treatment with 5 mM Cu2+ caused a reduction of 67% in the enzyme's chitinolytic activity. The recombinant protein had antifungal activity as revealed by its ability to inhibit the spore germination and mycelial growth of Penicillium herquei. The three-dimensional structure of VuChiI was resolved at a resolution of 1.55 A by molecular replacement. The refined model had 245 amino acid residues and 381 water molecules, and the final R-factor and Rfree values were 14.78 and 17.22%, respectively. The catalytic domain of VuChiI adopts an alpha-helix-rich fold, stabilized by 3 disulfide bridges and possessing a wide catalytic cleft. Analysis of the crystallographic model and molecular docking calculations using chito-oligosaccharides provided evidences about the VuChiI residues involved in sugar binding and catalysis, and a possible mechanism of antifungal action is suggested.
V. Looser et al., Appl Microbiol Biotechnol 101 3163 (2017).
As Pichia pastoris (syn. Komagataella sp.) yeast can secrete pure recombinant proteins at high rates, it is a desirable production system. The function of a novel synthetic variant of the AOX1 promoter was characterised comprehensively using a strain secreting Candida antarctica lipase B (CALB) as a model. A new time-saving approach was introduced to determine, in only one experiment, the hitherto unknown relationship between specific product formation rate (q p) and specific growth rate (mu). Tight control of recombinant protein formation was possible in the absence of methanol, while using glycerol as a sole carbon/energy source. CALB was not synthesised during batch cultivation in excess glycerol (>10 g l-1) and at a growth rate close to mu max (0.15 h-1). Between 0.017 and 0.115 h-1 in glycerol-limited fedbatch cultures, basal levels of q p > 0.4 mg g-1 h-1 CALB were reached, independent of the mu at which the culture grew. At mu > 0.04 h-1, an elevated q p occurred temporarily during the first 20 h after changing to fedbatch mode and decreased thereafter to basal. In order to accelerate the determination of the q p(mu) relationship (kinetics of product formation), the entire mu range was covered in a single fedbatch experiment. By linearly increasing and decreasing glycerol addition rates, mu values were repeatedly shifted from 0.004 to 0.074 h-1 and vice versa. Changes in q p were related to changes in mu. A rough estimation of mu range suitable for production was possible in a single fedbatch, thus significantly reducing the experimental input over previous approaches comprising several experiments.
S. Ohsawa, H. Yurimoto, Y. Sakai, Mol Microbiol 104 349 (2017).
Wsc family proteins are plasma membrane spanning sensor proteins conserved from yeasts to mammalian cells. We studied the functional roles of Wsc family proteins in the methylotrophic yeast Pichia pastoris, and found that PpWsc1 and PpWsc3 function as methanol-sensors during growth on methanol. PpWsc1 responds to a lower range of methanol concentrations than PpWsc3. PpWsc1, but not PpWsc3, also functions during high temperature stress, but PpWsc1 senses methanol as a signal that is distinct from high-temperature stress. We also found that PpRom2, which is known to function downstream of the Wsc family proteins in the cell wall integrity pathway, was also involved in sensing methanol. Based on these results, these PpWsc family proteins were demonstrated to be involved in sensing methanol and transmitting the signal via their cytoplasmic tail to the nucleus via PpRom2, which plays a critical role in regulating expression of a subset of methanol-inducible genes to coordinate well-balanced methanol metabolism.
S. Hosseinkhani et al., Photochem Photobiol 93 429 (2017).
Contribution of luciferin-regenerating enzyme (LRE) for in vitro recycling of D-luciferin has been reported. According to crystal structure of LRE, it is a beta-propeller protein which is a type of all beta-protein architecture. In this overview, reinvestigation of the luciferase-based LRE assays and its function is reported. Until now, sequence of LRE genes from four different species of firefly has been reported. In spite of previous reports, T-LRE (from Lampyris turkestanicus) was cloned and expressed in Escherichia coli as well as Pichia pastoris in a nonsoluble form as inclusion body. According to recent investigations, bioluminescent signal of soluble T-LRE-luciferase-coupled assay increased and then reached an equilibrium state in the presence of D-cysteine. In addition, the results revealed that both D- and L-cysteine in the absence of T-LRE caused a significant increase in bioluminescence intensity of luciferase over a long time. Based on activity measurements and spectroscopic results, D-cysteine increased the activity of luciferase due to its redox potential and induction of conformational changes in structure and kinetics properties. In conclusion, in spite of previous reports on the effect of LRE (at least T-LRE) on luciferase activity, most of the increase in luciferase activity is caused by direct effect of D-cysteine on structure and activity of firefly luciferase. Moreover, bioinformatics analysis cannot support the presence of LRE in peroxisome of photocytes in firefly lanterns.
M. P. Landry et al., Nat Nanotechnol 12 368 (2017).
A distinct advantage of nanosensor arrays is their ability to achieve ultralow detection limits in solution by proximity placement to an analyte. Here, we demonstrate label-free detection of individual proteins from Escherichia coli (bacteria) and Pichia pastoris (yeast) immobilized in a microfluidic chamber, measuring protein efflux from single organisms in real time. The array is fabricated using non-covalent conjugation of an aptamer-anchor polynucleotide sequence to near-infrared emissive single-walled carbon nanotubes, using a variable chemical spacer shown to optimize sensor response. Unlabelled RAP1 GTPase and HIV integrase proteins were selectively detected from various cell lines, via large near-infrared fluorescent turn-on responses. We show that the process of E. coli induction, protein synthesis and protein export is highly stochastic, yielding variability in protein secretion, with E. coli cells undergoing division under starved conditions producing 66% fewer secreted protein products than their non-dividing counterparts. We further demonstrate the detection of a unique protein product resulting from T7 bacteriophage infection of E. coli, illustrating that nanosensor arrays can enable real-time, single-cell analysis of a broad range of protein products from various cell types.
A. Madjid Ansari et al., Enzyme Microb Technol 98 67 (2017).
In this study attempts were made to investigate the effect of extremely low frequency magnetic field (ELF-MF) on expression of glucose oxidase gene from Aspergillus niger Z-25 in Pichia pastoris GS115 at optimal environmental conditions. GOX encoding gene from A. niger was expressed in P. pastoris GS115 employing methanol inducible AOX1 promoter. From resistant transformants to high zeocin concentrations, maximum GOX expressing transformant was isolated and used throughout the study. Optimal environmental conditions for achieving maximum glucose oxidase (GOX) production was determined by varying temperature (20-30 degrees C) and final methanol concentration from 0.25-1% (v/v). Maximum GOX production was obtained at 20 degrees C and 0.25% (v/v) final methanol concentration. Exposure carried out at extremely low frequencies ranging from 1 to 5Hz and the field strength of 30-100mT unceasingly. Biomass, GOX, protease productions and cell viability were examined in exposed and non-exposed groups every 12h up to 96h. Among different exposure conditions, the frequency of 2.8Hz significantly increased GOX production and cell viability, decreased non-specific protease activity and improved biomass production regardless of changes in field intensity. Overall, supported by results, P. pastoris exposure to ELF-MF in certain circumstances could effectively further increase maximum production of GOX as achieved by optimized preliminary environmental parameters.
K. Sakai et al., Appl Microbiol Biotechnol 101 3237 (2017).
A beta-1,4-mannanase, termed AoMan134A, that belongs to the GH 134 family was identified in the filamentous fungus Aspergillus oryzae. Recombinant AoMan134A was expressed in Pichia pastoris, and the purified enzyme produced mannobiose, mannotriose, mannotetraose, and mannopentaose from galactose-free beta-mannan, with mannotriose being the predominant reaction product. The catalytic efficiency (k cat/K m ) of AoMan134A was 6.8-fold higher toward galactomannan from locust bean gum, than toward galactomannan from guar gum, but similar toward galactomannan from locust bean gum and glucomannan from konjac flour. After incubation at 70 degrees C for 120 min, the activity of AoMan134A toward glucomannan decreased to 50% of the maximal activity at 30 degrees C. AoMan134A retained 50% of its beta-1,4-mannanase activity after heating at 90 degrees C for 30 min, indicating that AoMan134A is thermostable. Furthermore, AoMan134A was stable within a neutral-to-alkaline pH range, as well as exhibiting stability in the presence of a range of organic solvents, detergents, and metal ions. These findings suggest that AoMan134A could be useful in a diverse range of industries where conversion of beta-mannans is of prime importance.
Y. Zhang et al., J Ind Microbiol Biotechnol 44 453 (2017).
To achieve a cost-effective bioconversion of lignocellulosic materials, a novel xylose/glucose co-fermentation process by co-culture of cellulose-utilizing recombinant Saccharomyces cerevisiae (S. cerevisiae) and xylan-utilizing recombinant Pichia pastoris (P. pastoris) was developed, in which ethanol was produced directly from wheat straw without additional hydrolytic enzymes. Recombinant S. cerevisiae coexpressing three types of cellulase and recombinant P. pastoris coexpressing two types of xylanase were constructed, respectively. All cellulases and xylanases were successfully expressed and similar extracellular activity was demonstrated. The maximum ethanol concentration of 32.6 g L-1 with the yield 0.42 g g-1 was achieved from wheat straw corresponding to 100 g L-1 of total sugar after 80 h co-fermentation, which corresponds to 82.6% of the theoretical yield. These results demonstrate that the direct and efficient ethanol production from lignocellulosic materials is accomplished by simultaneous saccharification (cellulose and hemicellulose) and co-fermentation (glucose and xylose) with the co-culture of the two recombinant yeasts.
R. R. Prabhu, D. Parashar, T. Satyanarayana, Bioprocess Biosyst Eng 40 651 (2017).
The recombinant alkalistable and moderately thermostable bifunctional endoglucanase gene (BhCell-Xyl) of polyextremophilic bacterium Bacillus halodurans TSLV1 has been expressed in Pichia pastoris under constitutive GAP as well as inducible AOX promoters. A higher titre of recombinant BhCell-Xyl was attained after induction (4.8 U mL-1) as compared to that of the constitutive production (2.1 U mL-1). The recombinant P. pastoris strains integrated two copies of BhCell-Xyl under AOX and GAP promoters. The pure recombinant BhCell-Xyl is a glycoprotein of 66 kDa, which is optimally active at 60 degrees C and pH 6.0 and 8.0. Glycosylated BhCell-Xyl exhibits higher thermostability than that of the native enzyme. The analysis of amino acids of BhCell-Xyl revealed that multiple factors are responsible for its thermostability. Kinetics and in silico analysis of the enzyme suggested that BhCell-Xyl has one active site for both endocellulase and endoxylanase activities. The BhCell-Xyl possesses a carbohydrate binding domain and saccharifies lignocellulosic agro-residues to xylo-oligosaccharides and cello-oligosaccharides, suggesting its potential application in generating fermentable sugars from renewable agro-residues for biofuel and fine chemical industries.
J. Berrios et al., J Ind Microbiol Biotechnol 44 407 (2017).
The production of recombinant proteins by Pichia pastoris under AOX1 promoter is usually performed using methanol together with either glycerol or sorbitol as co-substrate. Although both co-substrates have been widely used, comparative studies are scarce. In addition, these comparisons have been performed at different specific growth rate (micro) that it is well known that has an important effect on productivity. Thus, the effect of using these co-substrates on the production of Rhyzopus oryzae lipase (ROL) by P. pastoris was compared in continuous cultures growing at the same micro at either 22 or 30 degrees C. Results show that using glycerol as co-substrate led to higher volumetric productivities, and lower specific and volumetric methanol consumption rates. Scale-up simulation with 10-10,000 L bioreactor sizes indicated that glycerol produced the highest volumetric productivity of ROL with lower aeration requirements. Therefore, glycerol rises as a better option than sorbitol in ROL production.
R. Chessa et al., Appl Microbiol Biotechnol 101 2931 (2017).
The use of natural antimicrobials from plants, animals and microorganisms to inhibit the growth of pathogenic and spoilage microorganisms is becoming more frequent. This parallels the increased consumer interest towards consumption of minimally processed food and 'greener' food and beverage additives. Among the natural antimicrobials of microbial origin, the killer toxin produced by the yeast Tetrapisispora phaffii, known as Kpkt, appears to be a promising natural antimicrobial agent. Kpkt is a glycoprotein with beta-1,3-glucanase and killer activity, which induces ultrastructural modifications to the cell wall of yeast of the genera Kloeckera/Hanseniaspora and Zygosaccharomyces. Moreover, Kpkt maintains its killer activity in grape must for at least 14 days under winemaking conditions, thus suggesting its use against spoilage yeast in wine making and the sweet beverage industry. Here, the aim was to explore the possibility of high production of Kpkt for biotechnological exploitation. Molecular tools for heterologous production of Kpkt in Komagataella phaffii GS115 were developed, and two recombinant clones that produce up to 23 mg/L recombinant Kpkt (rKpkt) were obtained. Similar to native Kpkt, rKpkt has beta-glucanase and killer activities. Moreover, it shows a wider spectrum of action with respect to native Kpkt. This includes effects on Dekkera bruxellensis, a spoilage yeast of interest not only in wine making, but also for the biofuel industry, thus widening the potential applications of this rKpkt.
G. Degani et al., Redox Biol 11 275 (2017).
The Advanced Glycation and Lipoxidation End products (AGEs and ALEs) are a heterogeneous class of compounds derived from the non-enzymatic glycation or protein adduction by lipoxidation break-down products. The receptor for AGEs (RAGE) is involved in the progression of chronic diseases based on persistent inflammatory state and oxidative stress. RAGE is a pattern recognition receptor (PRR) and the inhibition of the interaction with its ligands or of the ligand accumulation have a potential therapeutic effect. The N-terminal domain of RAGE, the V domain, is the major site of AGEs binding and is stabilized by the adjacent C1 domain. In this study, we set up an affinity assay relying on the extremely specific biological interaction AGEs ligands have for the VC1 domain. A glycosylated form of VC1, produced in the yeast Pichia pastoris, was attached to magnetic beads and used as insoluble affinity matrix (VC1-resin). The VC1 interaction assay was employed to isolate specific VC1 binding partners from in vitro generated AGE-albumins and modifications were identified/localized by mass spectrometry analysis. Interestingly, this method also led to the isolation of ALEs produced by malondialdehyde treatment of albumins. Computational studies provided a rational-based interpretation of the contacts established by specific modified residues and amino acids of the V domain. The validation of VC1-resin in capturing AGE-albumins from complex biological mixtures such as plasma and milk, may lead to the identification of new RAGE ligands potentially involved in pro-inflammatory and pro-fibrotic responses, independently of their structures or physical properties, and without the use of any covalent derivatization process. In addition, the method can be applied to the identification of antagonists of RAGE-ligand interaction.
G. Gupta, R. Glueck, N. Rishi, Biologicals 46 11 (2017).
There continues to be an urgent need for cost-effective prophylaxis for HPV-associated cancers in socio-economically underdeveloped nations. Presently HPV vaccines, which are commercially available, are adjuvanted virus-like particles (VLPs) expressed from various recombinant expression systems. They have been characterized by different methods as safe, pure, and potent HPV vaccine antigens. We cloned and expressed L1 proteins of HPV16 & 18 in Pichia pastoris and tested their immunogenicity. We observed that HPVL1 proteins (16L1 and 18L1) are expressed in Pichia pastoris at high levels. Critical physicochemical parameters of these HPV recombinant L1 proteins were characterized by SDS PAGE, western blotting, peptide mapping, glycosylation pattern, mass spectrometry, host cell DNA and protein analysis, electron microscopy, and immunogenicity analysis. These data establish a blueprint of HPV recombinant protein antigens for standardizing & developing an alternative high-quality, cost-effective vaccine for HPV as well as similar recombinant protein-based vaccines.
J. Aizen, L. Hollander-Cohen, M. Shpilman, B. Levavi-Sivan, J Endocrinol 232 391 (2017).
Currently, spawning is induced in carp species by carp pituitary extract (CPE) and a combination of synthetic agonist of GnRH combined with a dopamine antagonist. The main goal of this study was the production of recombinant gonadotropins (GtHs) on a large scale to serve as an alternative to currently used agents. We produced carp (c) recombinant (r) Lh as a single chain in the methylotrophic yeast Pichia pastoris Lha subunit was joined with Lhb subunit with a flexible linker of three glycine-serine repeats and six Histidines to form a mature protein, the beta-subunit formed the N-terminal part and the alpha-subunit formed the C-terminal part. The ability of the rcLh to elicit biological response was tested by in vivo stimulation of estradiol (E2) and 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP) and by its in vivo potency to induce ovulation and spawning induction. rcLh tested in this work significantly enhanced both E2 and DHP secretion in a dose-dependent manner similar to the results obtained with CPE. E2 levels showed a moderate rise following the priming injection and a subsequent decrease during the rest of the trial. DHP levels were only increased after the resolving injection, approximately 5 h before spawning. At the highest dose of rcLh (350 microg/kg BW), the recombinant protein was more efficient than CPE in terms of both spawning success and fertilization rate. It is shown here that rcLh can elicit the secretion of DHP in vivo and actually trigger spawning. These novel findings introduce the potential of utilizing recombinant gonadotropins in aquaculture.
S. Eissazadeh et al., Braz J Microbiol 48 286 (2017).
This study was carried out to express human epidermal growth factor (hEGF) in Pichia pastoris GS115. For this aim, the hEGF gene was cloned into the pPIC9K expression vector, and then integrated into P. pastoris by electroporation. ELISA-based assay showed that the amount of hEGF secreted into the medium can be affected by the fermentation conditions especially by culture medium, pH and temperature. The best medium for the optimal hEGF production was BMMY buffered at a pH range of 6.0 and 7.0. The highest amount of hEGF with an average yield of 2.27mug/mL was obtained through an induction of the culture with 0.5% (v/v) methanol for 60h. The artificial neural network (ANN) analysis revealed that changes in both pH and temperature significantly affected the hEGF production with the pH change had slightly higher impact on hEGF production than variations in the temperature.
R. M. Portela et al., ACS Synth Biol 6 471 (2017).
Synthetic biology and metabolic engineering experiments frequently require the fine-tuning of gene expression to balance and optimize protein levels of regulators or metabolic enzymes. A key concept of synthetic biology is the development of modular parts that can be used in different contexts. Here, we have applied a computational multifactor design approach to generate de novo synthetic core promoters and 5' untranslated regions (UTRs) for yeast cells. In contrast to upstream cis-regulatory modules (CRMs), core promoters are typically not subject to specific regulation, making them ideal engineering targets for gene expression fine-tuning. 112 synthetic core promoter sequences were designed on the basis of the sequence/function relationship of natural core promoters, nucleosome occupancy and the presence of short motifs. The synthetic core promoters were fused to the Pichia pastoris AOX1 CRM, and the resulting activity spanned more than a 200-fold range (0.3% to 70.6% of the wild type AOX1 level). The top-ten synthetic core promoters with highest activity were fused to six additional CRMs (three in P. pastoris and three in Saccharomyces cerevisiae). Inducible CRM constructs showed significantly higher activity than constitutive CRMs, reaching up to 176% of natural core promoters. Comparing the activity of the same synthetic core promoters fused to different CRMs revealed high correlations only for CRMs within the same organism. These data suggest that modularity is maintained to some extent but only within the same organism. Due to the conserved role of eukaryotic core promoters, this rational design concept may be transferred to other organisms as a generic engineering tool.
M. Nisha, T. Satyanarayana, Appl Microbiol Biotechnol 101 2357 (2017).
The 3'-deleted amylopullulanase gene from the extreme thermophile Geobacillus thermoleovorans (Gt-apuDeltaC) was expressed extracellularly in Pichia pastoris under both methanol-inducible AOX1 and constitutive GAP promoters. The expression of the gene (Gt-apuDeltaC) was higher under GAP promoter (36.2 U ml-1, alpha-amylase; 33.5 U ml-1, pullulanase) than that under AOX1 promoter (32.5 and 28.6 U ml-1). The heavily glycosylated Gt-apuDeltaC from the recombinant P. pastoris displays higher substrate specificity, thermal stability and starch saccharification efficiency than that expressed in Escherichia coli. The enzyme hydrolyses maltotriose and maltotetraose unlike that expressed in E. coli. The enzyme action on wheat bran liberates maltose and glucose without detectable amount(s) of maltooligosaccharides. The sugars released from wheat bran (glucose and maltose) could be fractionated by ultrafiltration, as confirmed by TLC and HPLC analysis. This is the first report on the production of recombinant amylopullulanase extracellularly in P. pastoris.
H. Yu et al., Dev Comp Immunol 68 108 (2017).
Defensins are a group of host defense peptides that play a central role in host innate immune responses. Here, 26 genes encoding beta-defensin-like peptides have been identified for the first time in Pelodiscus sinensis using database mining approach. Phylogenetic study confirmed that beta-defensins are fast evolving genes with high rates of sequence substitutions. The expression level of several selected genes in different tissues was examined by RT-PCR. Ps-BDs mainly adopt beta-strands and/or alpha-helix conformations homology modeled by Rosetta program. Further, Ps-BD2 was expressed in Pichia pastoris and purified using Ni-NTA column and RT-HPLC. As expected, the rPs-BD2 showed strong antimicrobial activity, but displayed a negligible hemolytic and cytotoxic activity on human erythrocytes and Raw 264.7 murine macrophage cells, respectively. Our results suggested that the Ps-BD2 was produced efficiently in P. pastoris expression system, which makes the large-scale use of rPs-BDs possible in the future clinical practice.
M. Wang et al., Arch Virol 162 701 (2017).
Herpes simplex virus type 2 (HSV-2) infection is the leading cause of genital ulcer disease and a significant public health concern. However, there are no approved vaccines available to prevent HSV-2 infection. The glycoprotein D (gD) of HSV-2 is the most important candidate antigen for vaccine development. In this study, a truncated form of gD (codons 1-340, gD1-340) was produced as a secretory protein in the methylotrophic yeast Pichia pastoris. The recombinant gD1-340 with a His6 tag was purified to homogeneity by one-step affinity chromatography. Mice immunized with the recombinant gD1-340 developed high levels of antigen-specific antibody responses with HSV-2 neutralizing activity. Immunization with the recombinant gD1-340 conferred significant protection against lethal HSV-2 infection in mice. Moreover, measurement of the secretion of gD1-340-specific cytokines demonstrated that the recombinant gD1-340 induced mixed Th1/Th2 cellular immune responses. These findings indicated that P. pastoris-derived gD1-340 represents a promising HSV-2 vaccine candidate with strong immunogenicity and prophylactic efficacy.
I. Effenberger et al., Appl Microbiol Biotechnol 101 2021 (2017).
We established an efficient fed-batch fermentation process for two novel dirigent proteins from cotton plants, GbDIR2 from Gossypium barbadense and GhDIR3 from G. hirsutum, using the engineered Pichia pastoris GlycoSwitch(R) SuperMan5 strain to prevent hyperglycosylation. The two (His)6-tagged proteins were purified by metal-chelate affinity chromatography and obtained in quantities of 12 and 15 mg L-1 of culture volume, respectively. Glycosylation sites were identified for the native and for the enzymatically deglycosylated proteins by mass spectrometry, confirming five to six of the seven predicted glycosylation sites in the NxS/T sequence context. The predominant glycan structure was Man5GlcNAc2 with, however, a significant contribution of Man4-10GlcNAc2. Both dirigent proteins (DIRs) mediated the formation of (+)-gossypol by atropselective coupling of hemigossypol radicals. Similar to previously characterized DIRs, GbDIR2 and GhDIR3 lacked oxidizing activity and depended on an oxidizing system (laccase/O2) for the generation of substrate radicals. In contrast to DIRs involved in the biosynthesis of lignans, glycosylation was not essential for function. Quantitative enzymatic deglycosylation yielded active GbDIR2 and GhDIR3 in excellent purity. The described fermentation process in combination with enzymatic deglycosylation will pave the way for mechanistic and structural studies and, eventually, the application of cotton DIRs in a biomimetic approach towards atropselective biaryl synthesis.
C. Jiang et al., Appl Biochem Biotechnol 182 261 (2017).
A novel beta-1,3-glucanase gene, designated Ccglu17A, was cloned from the biological control fungus Chaetomium cupreum Ame. Its 1626-bp open reading frame encoded 541 amino acids. The corresponding amino acid sequence showed highest identity (67 %) with a glycoside hydrolase family 17 beta-1,3-glucanase from Chaetomium globosum. The recombinant protein Ccglu17A was successfully expressed in Pichia pastoris, and the enzyme was purified to homogeneity with 10.1-fold purification and 47.8 % recovery yield. The protein's molecular mass was approximately 65 kDa, and its maximum activity appeared at pH 5.0 and temperature 45 degrees C. Heavy metal ions Fe2+, Mn2+, Cu2+, Co2+, Ag+, and Hg2+ had inhibitory effects on Ccglu17A, but Ba2+ promoted the enzyme's activity. Ccglu17A exhibited high substrate specificity, almost exclusively catalyzing beta-1,3-glycosidic bond cleavage in various polysaccharoses to liberate glucose. The enzyme had a Km of 2.84 mg/mL and Vmax of 10.7 mumol glucose/min/mg protein for laminarin degradation under optimal conditions. Ccglu17A was an exoglucanase with transglycosylation activity based on its hydrolytic properties. It showed potential antifungal activity with a degradative effect on cell walls and inhibitory action against the germination of pathogenic fungus. In conclusion, Ccglu17A is the first functional exo-1,3-beta-glucanase to be identified from C. cupreum and has potential applicability in industry and agriculture.
K. Melicherova et al., Appl Microbiol Biotechnol 101 1927 (2017).
Enterokinase is one of the most frequently used enzymes for the removal of affinity tags from target recombinant proteins. In this study, several fermentation strategies were assayed for the production of human enterokinase in Pichia pastoris under constitutive GAP promoter. Two of them with controlled specific growth rate during whole cultivation showed a very low enterokinase activity, under 1 U/ml, of the fermentation medium. On the contrary, the combined fermentation with a maximum specific growth rate at the initial phase of the fermentation and stationary-like phase during the rest of the fermentation showed a significant accumulation of the enterokinase in the medium, which counted up to 1400 U/ml. Lower cultivation temperature had a negative impact on the enzyme accumulation during this fermentation strategy. Downstream processes were focused on buffer environment optimization directly after cultivation, as at this time, the most amount of the activity is eliminated by endogenous proteases. Slightly positive effect on enzyme activity in the medium had an addition of liquid storage solution of EDTA and KOH to adjust pH to 8 and molarity of the EDTA to 50 mM. During the purification process, a significant amount of the enzyme was detected to be lost, which counted up to 90%. The purified enzyme, enterokinase, kept quality standard of the published enzymes.
M. Sigar, N. Maity, S. Mishra, Prep Biochem Biotechnol 47 364 (2017).
Protein fusion technology has emerged as one of the important strategies to increase the level of expression and half-life of therapeutic proteins in heterologous expression systems. Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor and is clinically used against neutropenia. Enhanced expression and stability of G-CSF were achieved in Pichia pastoris by the way of constructing a fusion protein with human serum albumin (HSA). The strategy involved polymerase chain reaction (PCR) amplification of fragments corresponding to codon-optimized G-CSF and domain 3 of HSA. Overlapping PCR was used to obtain the full-length fused gene (1,184 bp) with a 15-bp linker sequence comprising of 4 Gly and 1 Ser residues. Extracellular expression was carried out downstream of alpha-factor secretion signal sequence under the control of alcohol oxidase 1 promoter using pPICZalphaB. Excreted protein in the range of 110-380 mg L-1 was observed among the transformants. Effect of aeration and temperature was investigated in one of the transformants (35) overexpressing fusion protein and levels of G-CSF enhanced by 1.8-fold and 2.3-fold, respectively. Assay of biological activity indicated the fusion protein to retain similar cell proliferation activity as the commercial G-CSF preparation.
H. Faraji et al., Prep Biochem Biotechnol 47 379 (2017).
Staphylokinase (SAK) as the third generation thrombolytic molecule is a promising agent for the treatment of thrombosis. SAK variant of SAKsmall ef, CyrillicC was expressed in Pichia pastoris strains KM71H and GS115. The codon adaptation index of SAK was improved from 0.75 to 0.89. The expression of recombinant SAK (rSAK) reached to its maximum (310 mg/L of the culture medium) after 48-hr stimulation with 3% methanol and remained steady until day 5. The maximum activity of the enzyme was at pH 8.6 and 37 degrees C. It was highly active at temperatures 20-37 degrees C and pH ranges of 6.8-9 (relative residual activity more than 80%). It was determined that rSAK was 73.8% of the total proteins secreted by P. pastoris KM71H into the culture media. The specific activities of rSAK were measured as 9,002 and 21,042 U/mg for the nonpurified and purified proteins, respectively. The quantity of the purified protein (>99% purity) was 720 microg/mL with a purification factor of 2.34. Western blot analysis showed two bands of nearly 22 and 18.6 kDa. It was concluded that P. pastoris is a proper host for expression of biologically active and endotoxin-free rSAK due to its high expression and low protein impurity in culture supernatant.
S. Xiao et al., Prep Biochem Biotechnol 47 229 (2017).
Although the human antimicrobial peptide LL37 has a broad spectrum of antimicrobial activities, it easily damages host cells following heterologous expressions. This study attempted two strategies to alleviate its damage to host cells when expressed in Pichia pastoris using the AOX1 promoter. Tandem repeat multimers of LL37 were first designed, and secretion expression strains GS115-9K-(DPLL37DP)n (n = 2, 4, 6 and 8) containing different copies of the LL37 gene were constructed. However, LL37 tandems still killed the cells after 96 hr of induction. Subsequently, peroxisome-targeted expression was performed by adding a peroxisomal targeting signal 1 (SKL) at the C-terminus of LL37. The LL37 expression strain GS115-3.5K-LL37-SKL showed no significant inhibition in the cells after induction. Antibacterial activity assays showed that the recombinant LL37 expressed in peroxisomes had good antimicrobial activities. Then, a strain GS115-3.5K-LL37-GFP-SKL producing LL37, green fluorescent protein, and SKL fusion proteins was constructed, and the fusion protein was confirmed to be targeting the peroxisomes. However, protein extraction analysis indicated that most of the fusion proteins were still located in the cell debris after cell disruption, and further studies are required to extract more proteins from the peroxisome membrane.
M. Huang et al., Bioprocess Biosyst Eng 40 341 (2017).
Unfolded protein response (UPR) usually happens when expressing heterologous proteins in high level, which may help cells to facilitate protein processing. Here, we evaluated the effects of the UPR activator HAC1p on a raw-starch hydrolyzing alpha-amylase (Gs4j-amyA), so as to improve heterologous production of the enzyme in Pichia pastoris. The gene (amyA) encoding Gs4j-amyA was first codon-optimized and expressed in P. pastoris under the control of the AOX1 promoter. A high gene dosage (12 copies) of amyA facilitated amylase expression which produced an enzyme activity of 305 U/ml. A spliced HAC1 encoding an UPR activator HAC1p was then co-expressed and the dosage effects of HAC1 on amylase expression was investigated. Six copies of HAC1 driven by AOX1 promoter produced a high amylase activity of 2200 U/ml, further increasing by 621%. However, excessive gene dosages driven by the same promoter led to a titration effect of its transcription factors and decreased the amount of amyA transcripts. Thus, constitutive expression of HAC1 by GAP promotor was further involved and Gs4j-amyA activity reached 3700 U/ml finally, which was further increased by 68.2%. Moreover, Gs4j-amyA was glycosylated in P. pastoris which generated higher enzyme activity than that in E. coli. Generally, regulating HAC1p expression by different strategies enhanced amylase production by 11.1 folds, indicating a reference for expression of other proteins in P. pastoris.
F. Elahian, S. Reiisi, A. Shahidi, S. A. Mirzaei, Nanomedicine 13 853 (2017).
A genetically modified Pichia pastoris strain overexpressing a metal-resistant variant of cytochrome b5 reductase enzyme was developed for silver and selenium biosorption and for nanoparticle production. The maximum recombinant enzyme expression level was approximately 31 IU/ml in the intercellular fluid after 24 h of incubation, and the capacity of the recombinant biomass for the biosorption of silver and selenium in aqueous batch models were measured as 163.90 and 63.71 mg/g, respectively. The ions were reduced in the presence of enzyme, leading to the formation of stable 70-180 nm metal nanoparticles. Various instrumental analyses confirmed the well-dispersed and crystalline nature of the spherical nanometals. The purified silver and selenium nanoparticles exhibited at least 10-fold less cytotoxicity toward HDF, EPG85-257, and T47D cells than silver nitrate and selenium dioxide. These results revealed that the engineered Pichia strain is an eco-friendly, rapid, high-throughput, and versatile reduction system for nanometal production.
J. Li et al., J Biosci Bioeng 123 294 (2017).
A family 27 carbohydrate-binding module of a Thermotoga maritima beta-mannanase (TmCBM27) was chosen from the carbohydrate-active enzyme database by computer-aided design, possessing the lowest binding free energy with mannopentaose. To improve the enzymatic properties of a glycoside hydrolase family 5 beta-mannanase from Aspergillus usamii (AuMan5A), two fusion beta-mannanases, AuMan5A-F-M and AuMan5A-R-M, were designed by fusing a TmCBM27 into its C-terminus linked with a flexible peptide F (GGGGS)3 and rigid peptide R (EAAAK)3. Two fusion enzyme genes, Auman5A-F-m and Auman5A-R-m, were constructed as designed theoretically by overlapping PCR. Then, Auman5A and two fusion genes were expressed in Pichia pastoris GS115. Three recombinant beta-mannanases, reAuMan5A, reAuMan5A-F-M and reAuMan5A-R-M, were purified to homogeneity with specific activities of 230.6, 153.3 and 241.7 U/mg. The temperature optimum of reAuMan5A-R-M was 70 degrees C, identical with that of reAuMan5A, while its thermostability and melting temperature (Tm) reached 68 degrees C and 74.9 degrees C, being 8.0 degrees C and 8.4 degrees C higher than those of the latter, respectively. Additionally, the Km values of reAuMan5A-R-M, towards locust bean gum, konjac gum and guar gum, significantly decreased to 0.9, 1.9 and 2.5 mg/mL from 1.7, 3.8 and 4.2 mg/mL of reAuMan5A, while its kcat/Km (catalytic efficiency) values increased to 287.8, 163.7 and 84.4 mL/mgs from 171.2, 97.6 and 56.0 mL/mgs of the latter, respectively. These results verified that the fusion of a TmCBM27 into the C-terminus of AuMan5A mediated by (EAAAK)3 linker contributed to its improved thermostability and catalytic efficiency.
E. J. Kim, J. H. Lee, S. G. Lee, S. J. Han, Prep Biochem Biotechnol 47 299 (2017).
To survive in a subzero environment, polar organisms produce ice-binding proteins (IBPs). These IBPs prevent the formation of large intracellular ice crystals, which may be fatal to the organism. Recently, a recombinant FfIBP (an IBP from Flavobacterium frigoris PS1) was cloned and produced in Pichia pastoris using fed-batch fermentation with methanol feeding. In this study, we demonstrate that FfIBP produced by P. pastoris has a glycosylation site, which diminishes the thermal hysteresis activity of FfIBP. The FfIBP expressed by P. pastoris exhibited a doublet on SDS-PAGE. The results of a glycosidase reaction suggested that FfIBP possesses complex N-linked oligosaccharides. These results indicate that the residues of the glycosylated site could disturb the binding of FfIBP to ice molecules. The findings of this study could be utilized to produce highly active antifreeze proteins on a large scale.
W. Shen et al., Asian-Australas J Anim Sci 30 736 (2017).
OBJECTIVE: Experiments were conducted to clone the sequence of Wild Argali short palate, lung and nasal epithelium clone 1 (SPLUNC1) cDNA, and to lay the foundation for further study the biological function of Wild Argali SPLUNC1.
METHODS: The complete sequence of Wild Argali SPLUNC1 cDNA was generated by rapid amplification of cDNA ends. The entire coding sequence was inserted into the pPIC9K vector and expressed in Pichia pastoris (P. pastoris) GS115. The recombinant SPLUNC1 protein was detected by Western blot and purified by Ni2+ chelate affinity chromatography. The test of effect of the protein on Mycoplasma ovipneumoniae (MO) was performed with real-time polymerase chain reaction.
RESULTS: The Wild Argali SPLUNC1 cDNA was 1,076 bp with an open reading frame of 768 bp, which encoded a 26.49 kDa protein composed of 255 amino acids. Its amino acid sequence shared 98.4%, 96.9%, 94.5%, 90.2%, 80.8%, 78.4%, 78.3%, 72.5%, 72.3%, 68.8% identity with those of SPLUNC1 cDNA from Ovis aries (accession no. NP_001288334.1), Capra hircus (accession no. XP_005688516.1), Pantholops hodgsonii (accession no. XP_005979709.1), Bos taurus (accession no. NP_776851.1), Felis catus (accession no. XP_006929910.1), Homo sapiens (accession no. NP_001230122.1), Sus scrofa (accession no. NP_001005727.1), Chinchilla lanigera (accession no. NP_001269294.1), Mus musculus (accession no. NP_035256.2), and Rattus norvegicus (accession no. NP_742028.1), respectively. The recombinant protein corresponded to the expected molecular mass of 25.47 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and it was detected in the supernatant of P. pastoris, and it could be purified. The results from the test of inhibition effect of argali recombinant SPLUNC1 protein on MO showed that the product could inhibit MO very well (p<0.01).
CONCLUSION: The amino acid sequence of Wild Argali SPLUNC1 was different from other organisms. The recombinant SPLUNC1 protein has good biological activity.